Genetics chapter 7, 8

The Basics

- genes are transcribed -essentially the basic idea of transcription is to go from a DNA nucleic acid language (which is in long lived molecule) lives in the nucleus and is not subject to a lot of degradation we want to do is transcribe that into an RNA molecule which is a short lived nucleic acid that will then get exported into the cytoplasm of the cell. The reason we want to do this is so that the DNA molecules remain untouched and unharmed in the nucleus, by doing this type of transcription where we get DNA transcribed into a short lived RNA molecule we then essentially have a bunch of little copies of sequence that we can ship out into the rest of the cell. -And if they get degraded it's not as big of a deal as if the DNA were to become degraded. So the basics of transcription are that we are going to identify or region of DNA that encodes gene is a sequence of DNA that is transcribed. so a DNA sequence is just a DNA sequence and less actively undergoes transcription in which case we call it a gene. so in this case what you're seeing is one of the DNA molecules is going to be used as a template to create the complementary sequence in the RNA molecule, the template strand is also called the non coding strand, the reason for that is because the sequence that we're generating is going to be the complement to the sequence that we're using as the template. -So as the polymerase is going to make this new RNA molecule it's going to read the template strand but incorporate a complementary base sequence on the growing RNA molecules. So even though the template strand codes for ATGCC, the RNA molecule that we are making in the five prime to three different will be the complement of that so UACGG. The other strand in the DNA molecule is called the non template strand you can also call it the coding strand it will be the identical sequence of RNA that we're making, with the exception that we're exchanging T for U so you'll notice if you compare the RNA sequence to the coding strand sequence their identical which is why we call this strand the coding strand we do not use it to actually perform transcription we use the template strand but the sequence is useful because the sequence in the coding sharing is what sensually will be the sequence in RNA molecule that we're making

Functions of the Subunits

-Alpha subunit is going to have changed that are going to interact with regulatory proteins essentially this can either slow the function of RNA polymerase or it can increase the function of RNA polymerase. -Beta subunit, which is helpful in initiation and elongation, so this is going to be the part of the RNA polymerase that actually makes the phosphodiester bonds between adjacent nucleotides. -Beta prime unit which is helpful in the binding of the core polymerase to the DNA promoter element itself. -the Omega allows for the binding of the transcription factor which is called Sigma. -Then the sigma factor is going to help align this polymerase in its proper orientation such that we are going to start transcription at the plus one which is the proper place in promoter where we are going to start the transcription of the actual gene, -so all of these elements have different functions but they function collectively as one giant enzyme complex to carry out RNA polymerase function.

Termination

-And then we get into termination, this whole cycle multiple times then what happens is we should eventually will get to the Terminator sequence which is a stop codon. so here it is showing you in step one is our growing amino acid chain coming out the P site chimney once we get to a terminator sequence so a UAG,UAA, or UGA will center the A site over that stop codon, but there's no tRNA that corresponds to any one of those three sequences, So what happens is the ribosome kind of stalls out and can't do anything, and So what will bind instead of a tRNA to A site is what we call the release factor it is a little peptide that looks like the tRNA so it's very similar in shape it kind of wedges itself into the A site and then what it does is it causes a release of the tRNA from the polypeptide or the new amino acid sequence that we made. -So it releases the tRNA and the new protein that we've just made and then in step three what it does is it causes a small explosion inside the ribosome that causes the small subunit, large subunit, and mRNA to dissociate from one another. so it's like you planted a bomb in the A site and the A site kind of through hydrolysis of GTP causes a dissociation of the large subunit, small subunit, the release factor, and the mRNA and then essentially what happens is all of these components just get recycled again and we can make new protein from a different mRNA or the same mRNA doesn't really matter

Eukaryotic pre-mRNA Sequence transcribed from DNA

-In eukaryotes we first make a pre-mRNA sequence that is transcribed from DNA because we know that eukaryotes are going to process their mRNA molecule so we make a 5' mRNA we still have a 5' UTR and a 3' UTR but In addition to that we also have sequences within the quote protein coding region which is located in green we have two little orange sequences here or what we call introns. -so intron means interfering RNA in what those sequences are sequences that are eventually going to get cut out during RNA processing such that we string Exon1, Exon2, and Exon3 together. In addition to splicing of intron so that the exons are all uniform in back to back we also add a 5' cap to the 5' mRNA that's a little nucleotide that we flipped upside down so that it cannot be degraded in the cytoplasm. -And we add a 3' poly adenine tail. so we already looked a little bit about when that gets added which is during termination, but essentially with the 5' cap and polyA tail are going to so is prevent degradation of the sequence that we need in translation so they just prevent exonuclease activity from the 5' end and the 3' end

Polyribosome

-So what this is showing you is the Poly ribosome which happens both in prokaryotes and in Eukaryotes because essentially what happens is once we spit out a mature mRNA from the nucleus your eukaryote or just you know during transcription in a prokaryote. we can get multiple copies of translation happening just from one mRNA transcript. So what is showing you here is essentially we're going to bind the two ribosomal subunits to the 5' end at the mRNA and they're going to slide their way in this case from left to right, as they do that they're going to start making new polypeptides and new protein is gonna get spit out of the chimney of each one of these ribosomes and we can have what's called a Poly ribosome -so we're manufacturing multiple copies of the protein from just one mRNA which really speeds up the process of protein manufacturing in both prokaryotes and eukaryotes. so you make 1 mRNA copy but you can be making lots and lots of copies of the protein just from that one transcript, and you can actually physically see this under an electron micrograph so here what it showing you in panel B is that you have one along like ribbon of mRNA and then you can see the little ribosome manufacturing facilities that are located all along the ribbon of mRNA so we're actually producing a crap ton of protein from this one mRNA transcript and that will continue to occur until the mRNA is degraded

The Basics Where in the cell does transcription occur?

-so on the left on this slide showing you is arrangement of the DNA molecule in a cell that does not have a nucleus so remember prokaryotes they do not have membrane bound organelles. Which includes the nucleus, so in prokaryotes the DNA is just kind of floating around in the cytoplasmic soup it's not protected by a nuclear envelope and thus we can perform transcription and translation together. -So the process of transcription is occurring in the cytoplasm as is the process of translating that RNA molecule that we were making into proteins. So we have our DNA molecule we have our RNA polymerase that's going to make our mRNA copy, and then as soon as we start to make that mRNA copy our ribosomes can bind to the mRNA molecule and start firing off copies of the protein through the process of translation. This is called Collinearity of expression all that means is that essentially transcription and translation occur simultaneously this allows the prokaryote to adapt very quickly to its surroundings, but it doesn't allow the prokaryote to change any of the sequence of mRNA that's being made before it's translated so literally there is no time to pause or stop or process the mRNA that's being made in the prokaryotic cell. -Conversely eukaryotes have their DNA protected in this nice little nuclear envelope so the DNA exists in this nice sheltered environment in the nucleus and then so transcription is going to occur inside the nucleus and it is physically impeded from becoming translated until it's until an mRNA molecule is processed and shipped out into the cytoplasm so there is a physical barrier between where M RNA transcription is occurring and where translation is occurring so they do not occur in tandem like they do in prokaryotes. -We actually stop the process and then we process our mRNA so we add a 5' cap we add a Poly a tail, and we splice out any intron sequences in the nucleus and then once all of that is done then we can ship out the mRNA into the cytoplasm where we can produce our amino acid sequences and we combined are ribosomes to make these new protein molecules.

Characteristics of an mRNA gene -- Eukaryotes

-we have some additional sequences in eukaryotes that we need to be aware of so just like in prokaryotes we have three major parts of the gene architecture. We have the promoter which is the same as in prokaryotes although the sequences that we care about are going to be located at slightly different places within the promoter, so we have our plus one that extends through the Terminator sequence, and then within the RNA coding sequence we have what we call exons and introns. So here you see your first ATG which is going to be the translation start site after that we have Exon-one, intron- one, exon two, intron-two, Exon 3, and then the stop sequence followed by the Terminator. -When we produce our pre-mRNA molecule we are literally going to include everything between the plus one in the end of the Terminator sequence, including the introns. Eventually once we made our pre-mRNA molecule we're going to chop out those introns sequences, such that we only have the plus one, the translation start sequence which is the ATG, Exon one, then Exon two, then Exon three, stop sequence. So eventually those will be cleaved out of that sequence during processing and that will allow us to produce the fully mature mRNA. If you look back at the promoter you'll also notice we have not labeled the -10, we have labeled the -25 and the -35 these are two special sequences within the promoter of the mRNA genes in eukaryotes. But they function very similarly to the pribnow box that we find appropriate at the -10. -The TATA box is very similar to the pribnow box, and then the CAAT box is a little bit further upstream that is going to be helpful during initiation. In addition to just the regular core promoter which is labeled in those kind of Jade green color, a little bit upstream of that we have something called upstream enhancers and silencers this is in the upstream promoter so we can actually extend the promoter sequences in eukaryotes and this gives a level of sophistication to the actual gene expression system in eukaryotes that prokaryotes don't have access to. -There are a couple of additional elements that are found in the eukaryotic structures, so we have enhancers and silencers, we have two different types of sequences within the core promoter, and then actually we have some different sequences in the RNA coding sequences meaning exons and introns and then if you look at the Terminator we also have two additional sequences that are not in prokaryotes, we have polyA signal and we have the termination sequence found within the Terminator. it is a very good idea to go ahead and make yourself a table that compares prokaryotes to eukaryotes and the architectural sequences found within regular core promoter, the RNA sequence, and the terminator.

Central Dogma of Cell Biology

-so we're going from the DNA sequence through transcription if any transcriptional processing happens that's where we get our mature RNA molecule that will then undergo the process of translation so the lecture or for this particular module is going to focus on what happens in the transformation of RNA to protein in a process called translation. Which will then ultimately result in the function of the particular protein of interest and that of course will have impacts on the phenotype or the actual function of the protein that we manufacture so that's where we are in talking about the central dogma of this process

The Basics—General process overview Tools for transcription BOTH prokaryotes and eukaryotes:

1.DNA sequence (gene) elements -Signal sequences for initiation and termination -Template sequence for elongation -the tools transcription in both prokaryotes and eukaryotes we will need is a DNA sequence or a gene element so this is going to be the sequence of DNA that's required for serving as the template to make our new RNA molecule, we will have several signal sequences within that gene sequence we will have an initiation sequence and termination sequence, and of course between those two sequences we will have the actual sequence that needs to be copied that will serve as the template for the actual mRNA sequence that we're making. 2.Protein machinery -RNA polymerase -Transcription factors -We will also have some protein machinery will have what's called RNA polymerase this is basically the enzyme that's going to string those triphosphate nucleotides together form a new file phosphodiester bond and will have some additional proteins called transcription factors that will help us organize the whole entire process 3.Other ingredients -Ribonucleotides (NTPs: ATP, GTP, CTP, UTP) -we also need some other ingredients like the ribonucleotides NTP's we are not going to use TTP so remember we don't use thymine in RNA only Uracil

Central Dogma of Cell/Molecular Biology

DNA is the master instruction sequence Is transcribed into RNA (transient molecule) rRNA & tRNA (functional RNAs); mRNA (translated) Short-lived mRNA is translated into amino acid sequence to make protein -revisiting the central dogma of cell or molecular biology whereby we have our DNA that's our heritable molecule of inheritance. Big green arrow is showing you the process of replication which is the ability to replicate DNA and then pass it on To offspring cells and now we're going to do is talk about the rest of this schematic so talk about how DNA is expressed through the process of transcription to make RNA molecules. And then how we undergo the process of translation to produce proteins that eventually function to give the cell its particular phenotype, so here is a nice little schematic of the types of RNA products that are going to be made from the process of transcription. -Essentially what we're going to do here is exchange the DNA nucleic acid language for RNA nucleic acid language that will get us all of our functional RNA's In addition to the template RNA that we're going to use to translate that sequence into protein So we will make three major types of RNA's from these gene sequences will talk about tRNA, rRNA, mRNA. in all three of these function within the ribosomal protein machinery to then undergo the process of translation whereby we will get M RNA translated into protein, that protein will have some function and then that function will confer some phenotype for the cell

How are these three parts arranged within a gene?

Structure of a gene Differences between types of genes

mRNA Transcription Termination in Eukaryotes

Termination Involves Rat-1 Endonuclease + Complex of Proteins

Poly A proteins stabilize the structure

With eukaryotes is that the Poly a tail in the 5' cap can coordinate the stabilization of the structure of mRNA binding to the ribosome so essentially not only are the 5' cap and 3' polya tail helpful in the prevention of degradation of the mRNA's in eukaryotes. But they actually physically help stabilize the ribosome EIF initiation of translation in eukaryotes

Small RNAs: miRNA and siRNA -These RNAs regulate the translation of other mRNAs

miRNA Processing 1.RNA forms a hairpin through inverted repeats 2.Loop cut off hairpin 3.DICER chops sequence into pieces 4.Single stranded pieces loaded into RISC complex 5.RISC+miRNA forms complementary pairing with mRNA (imperfect pairing) 6.Inhibits translation; limits protein production siRNA Processing 1.Single stranded pieces loaded into RISC complex 2.RISC+siRNA forms complementary pairing with mRNA 3.Inhibits translation; limits protein production (perfect pairing) 4.leads to mRNA degradation -Both of these types of RNA molecules are going to undergo a tremendous amount of processing. The series of processing steps is very similar for both micro RNA and siRNA, the function of each one of those is slightly different though. So they're both going to inhibit translation of an mRNA molecule that has already been transcribed but micro RNA or and miRNA is going to reversibly inhibit translation. Where siRNA is going to lead to degradation of the mRNA altogether so it is physically going to not only stop a translation of this mRNA but it's going to get rid of the mRNA molecule altogether so it is irreversible we cannot reverse this process. -So first thing that happens with the micro RNA is we transcribe it when we transcribe it this molecule has complementary anti parallel bases in the sequence and so when we make this micro RNA it forms the stem loop structure where we have a piece of double stranded RNA and we have this moving that kind of turns the molecule around, this is called a hairpin. What we do next with it is we take the hairpin part off of the double stranded RNA so essentially what is going to happen is we chop off that little looping area that connects the two parts of the folded RNA molecule. The enzyme called dicer is then going to chop the complementary folded sequence of that stem part into multiple pieces of double stranded RNA, then we're going to do is choose at random some of those sequences that we have chopped and we're going to load single stranded parts of that micro RNA sequence into a protein complex called risk. -The risk complex is going to essentially have a little cavity where the micro RNA sequence is held in position and then what is going to happen is that risk complex with the micro RNA in it buried inside the protein is going to be able to bind through complementary base pairing to mRNA that has already been manufactured by the cell. Usually this base pairing is not perfect so some of the bases will bind but it won't be 100% complementarity. when this happens it stops the mRNA from being able to be translated into protein so it is functionally inhibiting the translation of this molecule. this is one level of regulation of gene expression for this particular mRNA. so maybe the cell decided it wanted to do gene expression of this mRNA but maybe now it has too many copies of that mRNA and it wants to kind of slow down the process of making the protein. -So what it does is it just basically reversibly inhibits the translation of this mRNA molecule to kind of slow down the process of generating too much protein. If the cell decides that it wants more protein all it has to do at this point is remove the risk complex from this mRNA and we can fire off more copies of the protein. So it's a reversible type of translation incubation. Conversely in siRNA processing you get similar situation so you produce double stranded silencing RNA's, one of those molecules gets loaded into the risk complex just like we see in micro RNA, that forms a perfect base pair with some sequence on some mRNA. what happens then is that leads to degradation of the mRNA molecule so not only are we pausing translation in silencing we are physically causing the degradation of that mRNA molecule. -so that is a permanent way of regulating our gene expression in eukaryotes. So micro RNA is a temporary slowdown of the process of translation, siRNA is a permanent reduction of the amount of protein that we are making from the mRNA molecule. so these are both ways to inhibit the process of translation miRNA is reversible , siRNA is irreversible

Pre-mRNA to Mature mRNA Processing Eukaryotes only Pre-mRNA Processing in Eukaryotes

•5' cap - during elongation (co-transcriptional) •Poly-A tail - immediately after termination (post-transcriptional; during termination) •Splicing/removal of introns - before exit to cytoplasm (post-transcriptional) -last thing after we add the cap in the tail that ribbon of mRNA is still existing in the nucleus but it is now not associated with the DNA and it is now not associated with the RNA polymerase. so it is freely floating in the nucleus we've added the cap, we have added the tail post transcriptionally, and then the last thing we need to do is splice the introns out of the protein coding sequence before we can ship this molecule out into the cytoplasm

Parts of Translation

•Before translation begins—charging of the tRNAs using aminoacyl tRNA synthetase (continues throughout translation process in the background of cell) •Initiation •Elongation •Termination •**Sound familiar? -Before we're going to start translation we just charge all of the tRNA using the aminoacyl tRNA synthetase that just happens all the time. so just think about like we're constantly turning over tRNA's and adding new amino acids in the charging process and then basically like during the process of translation we just we just pull from the pool of charged tRNA. so as always we have three parts of this process we have initiation innovation in termination. we will do a comparison of criteria to be periods but really the only difference is found in initiation

Alternative Splicing

•Introns allow eukaryotes to produce different isoforms of proteins from a single gene sequence •Makes isoforms by selectively splicing out some exons during intron removal (removes protein domains that regulate function) •Each isoform has a unique function -so you might be asking yourself what is the point of generating A mRNA molecule that has a bunch of introns that we just have to splice out like why on earth would a eukaryotic arrange it's genome such that we have a bunch of garbage sequence that we need to get rid of in the next process. so the reason that eukaryotes arrange their gene this way is it provides them flexibility in production of the final protein. so you can have what's known as alternative splicing or alternative removal of specific introns from the same mRNA molecule which will change the function of the protein that you are going to generate through the process of translation. -So introns are basically going to give flexibility to eukaryotic cell to produce a slightly different protein from one specific gene sequence. so like a brain cell can produce protein A or this version protein A from this gene and a blood cell can produce protein B which is a slightly different isoform from the same gene sequence. In these two proteins will have radically different functions but they were encoded for on the same gene. So it saves room in the genome. So when we make alternative proteins these are called isoforms, which means they it implies that they come from the same gene but they are the product of different spicing patterns. -So in this particular gene the DNA has 5 different exons and four different introns. Each Exon is going to encode a different part of the protein known as a domain, so the full expression of this protein is protein a where we have Exon1, Exon2, Exon3, Exon4, and Exon5 and we've removed all four of those introns. So essentially what we've done is after splicing we put Exon 1, 2, 3, 4, and five together in the sequence that gives us protein A after the process of translation which has five domains. It has domain number one which is a series of alpha helices, domain number two also a series of Alpha helices, domains three and four are some looping domains and then domain #5 is another Alpha Helix. -so maybe we want to produce the entire functional protein, protein A if we do that we're just going to splice out each intron individually so each intron will get cut at the 5' end it will get looped around two internal branch point, then it will get cut at the 3' end and then the two adjacent exons will get spliced together. So this will happen for the region between Exon1 and 2, the region between Exon2 and 3, region between 3 and 4, regions btw 4and 5. All of those exons will get spliced together but let's say we're a different type of cell and we don't want to have domain #3 in the final protein that we make, so we want to make protein B which does not include Exon 3. -What we will do is splice the 5' end of the second intron that's at the border of Exon 2, we will loop that 5' end of intron 2 over Exon3 to meet the branch point in intron 3 or the intron between Exon 3 and 4, and then we will cut the 3' end of intron 3 such that the 3' of Exon 2 and 5' end of Exon 4 get ligated together, and the lariat that's form that will be degraded includes intron 2 and Exon 3 and intron 3. So we've effectively spliced out not only the two introns that border Exon 3 but also Exon three. so we're not going to get that domain in the final protein because we've thrown it away. so protein B then will include Exon1, Exon 2, Exon4, Exon 5 but it will not include Exon3 which codes for that first loop domain. -Same thing with protein C in this case we don't want to contain the loop domain and Exon 4 so we just splice over it and we just take intron 3 splice that to the branch point in intron 4 and then Exon3 and Exon5 were gonna get ligated together and Exon4 is going to get thrown out in the lariat between intron3 and intron4. so basically it's just giving us a lot more flexibility with the type of proteins that we're producing such that we can produce in this case 3 different proteins from one gene sequence. so depending on what cell when dealing with and what the function of the cell in the tissue is we can produce radically different proteins that have completely different functions from one single gene that is the benefit of alternative splicing

Topic Outline

•Review central dogma •Features of the universal code •Elements of translation •tRNA and charging •Ribosome structure •Sequence of events •Initiation •Elongation •Termination •Impact of point mutations

Elements of Translation

•What are the major players in translation? •mRNA sequence (w/ codons) -Start and stop sites •tRNAs •Ribosomes (protein and rRNA) •ATP and GTP •Amino acids •Amino acyl synthetase

Cleavage of pre-mRNA and Synthesis of poly A tail

-Essentially when we do that initial cleavage Image site during termination in eukaryotes we also add the Poly a tail which is going to protect the 3' end of the new mRNA molecule that we have. so this is specifically a processing that occurs of the mRNA that happens in eukaryotes but do not happen in prokaryotes. -I want to point out is just how this is happening during termination so at the bottom of this figure you have your DNA molecule you have your RNA polymerase which is just now transcribing garbage RNA sequence what is circled is the 3' end of the pre mRNA that you have made where it was originally attached to the RNA that is being made after termination so where it says cut and it has two arrows within that red circle, those two pieces used to be attached. -so the piece that is attached to the RNA polymerase used to be attached to the 3' site of the pre mRNA what happens is during termination we cut that 3' site and immediately add in PolyA tail to the 3' end. so two things happen we cut beside the add the PolyA tail to that site and then rat1 is going to bind to the remainder of the RNA that's being produced by the RNA polymerase and will chew it until it is terminated. in the meantime everything that's connected to this pre mRNA with the Poly a tail is going to undergo some more processing in the nucleus and then will get shipped out into the cytoplasm so that it can be translated

Chromatin Remodeling Complex - Eukaryotes

-In Eukaryotes we tend to incircle the DNA molecules around the organizational proteins to form these nucleosomal balls that are connected by linker DNA. If your promoter element is found in In complex with one of those histone protein complexes then how on earth are you going to start the process of transcription. The answer is a protein complex called the chromatin remodeling complex, which is exclusive to eukaryotes. -So in the figure what it showing you is that we have region of DNA that we want to transcribe but the Tata binding site or the TATA box (in green) in the promoter is actually in complex with one of those histone octamers, so it's part of the nucleosome. So if that sequence is part of the nucleosome we cannot start transcription there, but we have to do is make sure that TATA binding site is found in one of the linker DNA regions. -So what we can do if we're a Eukaryote and we need to turn on gene transcription from a gene where the TATA binding site in a promoter is found in complex with one of these nucleosomes, is we can take the chromatin remodeling complex and kind of swivel the nucleosomes, such that we move the parts of the DNA that are interacting with the histones into a different orientation. So what is showing you in panel B is that chromatin remodeling complexes is kind of swiveling or spooling the nucleosomes around the DNA so they are kind of swiveling the position of the DNA that's interacting with the protein, such that we can move the TATA binding site into a region of linker DNA. -So we are moving it off of one of those histone molecules and into the linker region. When we do that now the DNA is free to bind to all of these transcription factors to the RNA polymerase, and then what we can do is once the RNA polymerase binds to that region of linker DNA as the RNA polymerase slide down to the right to start incorporating new RNA nucleotides, it will just shove this histone out-of-the-way. It's very similar to the process that we see in DNA replication where we can actually just like shift the sequences that we need to access off of histone complex when we need to and then re-spool them around a histone complex once the process has been completed. -So it's never really possible to have just completely naked DNA like that doesn't happen, it's always at the very minimum organized into these nucleosomal subunits. So the way that we get around any sort of problem with that where we have TATA binding site not in the optimal position to start transcription, is we just use that chromatin remodeling complex to kind of swivel the sequences around such that we could put them in the linker DNA segment.

Initiation of Transcription - DNA signal sequences

-Initiation sequences are ALWAYS found on the coding (non-template) strand -the first phase of transcription is called initiation and essentially the part of the DNA that's really central to performing initiation of transcription is the promoter. so here is an example of what the general architecture of mRNA gene looks like in prokaryotes. what is yellow orange color is the actual promoter and then it showing you in the bottom panel what the sequences are going to look like within that promoter panel, so it's actually like a blowup of this. -So in the promoter what we're seeing is that the minus ten position which means 10 nucleotides upstream in this case to the left of the plus one site we have that pribnow sequence which is called TATAAT it is always the same and it is always found on the coding strand also known as the non template strand this is very important for when we start actually doing transcription. -So the strand that is going to be used for the template to make the new sequence of RNA in this case the bottom strand, So the pribnow box the TATAAT sequence is found in the other strand so on the top strand in this case or the coding strand, additional promoter elements that are helpful at getting RNA polymerase to recognize this as a region Of mRNA gene sequence, can also be this minus 35 consensus sequence because it's always found at -35 position so approximately 25 nucleotides further upstream from the pribnow box we find this TTGACA sequence also called the minus 35, you don't need the minus 35 but you do need minimally the pribnow box at minus 10. -Then we're going to do is aggregate all of our RNA polymerase subunits onto this promoter with the exception of the Sigma factor, so the single factor is not going to bind until we're absolutely ready to start transcription. The core enzyme will bind to the promoter and it will centralize its finding right on top of this pribnow box and the reason that it does that is because the size of the polymerase is approximately the same size as 10 nucleotides. -so if it centers itself over the pribnow box the region where we're going to start incorporating new nucleotides will be centered right above the plus one, which conveniently is the transcription start site. so if you move this pribnow box a little bit further upstream or a little bit downstream it will throw off the whole frame of where we're going to start incorporating these nucleotides so the positioning is very important in the promoter.

How does a spliceosome recognize an intron and remove the correct sequence?

-Introns have distinguishable sequences -So the way that we physically do this how we physically trim out an intron from between two exons is dependent on three specific gene sequences. you have Exon1 in this case on the left hand side intron in between and then Exon2 on the right hand side. in the region or in the border between Exon1 and the 5' end of the intron you have what's called the 5' consensus sequence or the 5' splice site. -the sequence is always the same it literally exists on the border between the Exon in the 5' end of the intron. In addition to that you have a similar site on the 3' end of the intron next to the Exon2, so the 3' consensus sequence or the 3' splice site exists in the border between the 3' end of the intron and the 5' end of the Exon number 2. and then between these two sequences you have what's called the branch point, the branch point is always an adenine and it is located at a similar location within the Intron in this case its kind of 2/3 the way through the intron. -these sequences are going to work together with the spliceosome to inform the spliceosome where the looping in the intron is going to occur and where the splicing at the 5' end and the 3' end are going to occur. so this is very important these are your DNA signal sequences within the intron and exon sequences that will tell the spliceosome where things need to be moved

Specific Promoter Signal Sequences--Eukaryotes

-Just like in prokaryotes here is a slide of a blow up of what the promoter is going to look like in the different elements that are located within the core promoter. Not all of these elements are going to be there all of the time sometimes their positions are a little bit different, so the ones to specifically know are the TATA box located at -25. -The TFIIB recognition element that one is always present but not always present at the -35, so sometimes that position will shift a little bit. Then of course you always have your +1, and then sometimes you have both upstream and downstream promoter elements, so it's showing you in the figure is you have a downstream promoter element which is at the +30 position that's actually into the sequence that you're going to be transcribing, that one is not always there sometimes it's there. -And then you can also have an upstream regulatory which is found really far upstream of the core promoter, so this is usually hundreds base pairs to thousands of base pairs upstream of the core promoters. Basically these two types of promoter elements are present in a lot of genes but not all genes so they're not universal it usually has to do with the gene, when we want to turn the gene on versus when we want to turn the gene off, so it allows Eukaryotic cell to kind of fine turn their gene expressions in a easier and more sophisticated way than with prokaryotes

Termination Sequences and the Rat1 protein

-Just like in rho dependent termination in prokaryotes rat1 termination also involves a protein that is going to physically stop the RNA polymerase from transcribing any further. Eukaryotes though we don't have those convenient anti parallel complementary bases that will get formed in the mRNA sequence. -So there's no hairpin that forms in the termination sequence but what we do have is called a cleavage site and then in rat1 endonuclease binding site. So starting in the top left of this figure we are making our knew mRNA from the RNA polymerase which is scooting its way down to the right in this case what's circled in red here and shown with an arrow is what we call the cleavage site so the cleavage site is found within the Terminator sequence and causes cleavage of the mRNA molecule away from the RNA that's being transcribed following the Terminator. -so we're going to clip that mRNA at the cleavage site and then that mRNA molecule goes to be processed and translated in the nucleus and then again in the cytoplasm. So that mRNA molecule on the left hand side that we've clipped off of the DNA RNA polymerase complex will go away and it will continue its processing and its translation. What we're left with is essentially a runaway RNA polymerase so even though we have a Terminator sequence even though we're supposed to stop transcription at the site determination the RNA polymerase cannot help itself it cannot stop itself it just keeps going until we have that rat1 endonuclease stop the process. -After we do cleavage at the cleavage site we have a new 5' end that is attached to the RNA polymerase which is attached to the DNA molecule but this is all nonsense RNA and it is not RNA that we're going to use to make new proteins it is just garbage sequence it's meaningless to the cell. So we have to get rid of that meaningless mRNA like molecules. So what happens is after we cleave that site the rat1 endonuclease binds to the new 5' end of the garbage RNA molecule that's continuing to be synthesized and it chases the RNA polymerase just like rho does in prokaryotes, but now rat1 has an additional capability which is that it has 5' to 3' exonuclease activity. -So even though we call it a rat one endonuclease it has exonuclease activity as well which means it's going to digest the RNA that's being synthesized by the RNA polymerase from the 5' and toward the 3' so it binds to the new 5' site after cleavage happens and then as it chases the RNA polymerase it choose off that 5'site nucleotide by nucleotides it keeps on chewing the way I like to think about this is it's a rat, and it chews things. so the rat one chases the DNA sorry the RNA polymerase as its synthesizing it's degrading from the other end and then once it catches up to the RNA polymerase it unravels the whole complex so it kicks off the RNA polymerase from the DNA molecule it digests the rest of the garbage RNA sequence and then everything returns to normal

Products of transcription from rRNA and tRNA genes

-No we're going to talk about the gene arrangement and the products of transcription where the two other types of RNA's that we really care about so the product of transcription for rRNA and tRNA are going to just be like little functional folded RNA molecules. so the mRNA molecules that we make do not get folded into a crazy secondary structure with the exception of the hairpin that forms during termination of prokaryotic transcription we pretty much just get a long ribbon of mRNA. -tRNA and rRNA form these beautiful 3 dimensional structures through complementary base pairing because they are functional RNA molecules. so they're not just a template to translate into amino acid they're actually going to play roles during translation but they themselves are functional so they're functional RNA's. What I want to point out to you here is that the architecture of the gene setup is different for rRNA and tRNA genes than it is for mRNA genes. -so in prokaryotes they tend to group both rRNA and tRNA genes under a single promoter, so we'll actually make a long ribbon of RNA and then it'll get chopped into its individual components. so in this top panel what I'm showing you is where we have orange blocks those are going to be internal spacers or promoters, and so essentially that is where we're going to start our transcription. so at the terminal left end of this particular gene you'll make your 16S rRNA you'll make 2 tRNA's in this dark green color within the internal spacer you'll then make a larger 23S rRNA then you can make 5S rRNA and then two more tRNA so essentially this whole thing gets made as one long RNA molecule and then chopped into its individual components by use of these internal spacers. -in eukaryotes we do group rRNA's but we do not group tRNA. so tRNA's are made independently and so here you have an external spacer in 18S RNA an internal space are small RNA at another internal space are a larger RNA and then an external spacer. So what we can do is transcribe all of this is one long thing and then cut it at the spacer regions

Ribosomes

-Now we're going to do is talk about the ribosomes, so the ribosomes are going to be the actual physical machinery that's required for putting together the tRNA bringing in the amino acid with the mRNA these are going to be actual like little factories that are going to physically build the amino acid chain that physically make the protein. So the two major components that we have to talk about are at the small subunit of the ribosome and the large subunit of the ribosome. -So what is showing you here is with a small subunit has this little mRNA binding site, and basically what we're going to do the thread the mRNA through the junction between the large subunit and the small subunit. So basically where those subunits are into contact with one another, that is where the mRNA is going to sit. so it's kind of like of the sandwich. And we have three major binding parts of the large subunit so there are three binding sites. there's the E site which stands for the exit site, there is the P site which is the peptidyl-tRNA binding site which has this chimney that comes out of the top of the large subunit, and then we have the A site which is where we're going to bind the new tRNA that are carrying the amino acid

Prokaryotic mRNA Sequence Transcribed from DNA

-Processing of prokaryotic mRNA is unnecessary due to colinear translation -No pausing between transcription and translation; no processing of mRNA -prokaryotic mRNA sequence that's transcribed and we have a 3' end, we have a leader sequence in a trailer sequence, and then we have this protein coding sequence found within or between the to. Draw your attention to the start site for translation which is our next process that we're going to do is not the same as the start site for transcription, which would have been the +1 site on the 5' app. -so we have our translation and all of the sequence between the start site and the end site is used in the protein coding sequence for translation. there is no pausing between transcription and translation so the 2nd this mRNA starts to be generated we can actually start to do translation. so we couple these two processes there is no modification of this mRNA when it's being made in the process of transcription

Characteristics of genes coding for rRNA and tRNA-Prokaryotes

-Produce tRNA and rRNA together! Regulated under a single promoter—Makes a single polycistron! -Here is nice close up of what an rRNA-tRNA gene is going to look like inside prokaryote. so we're gonna make them together and we're gonna regulate them under a single promoter. so in this slide you see your little blue element on the left hand side and then this dark green element that is going to be the promoter for all of these genes. -so you have a 16S rRNA, 2 tRNA's, 23S rRNA, 5S rRNA and two more tRNA's genes before you reach a Terminator. so you'll make this whole thing is one long giant rRNA-tRNA and then you will chop it into its individual components once you're completely done with the transcription

Template strand determined by location of DNA signal sequences

-Promoter is ALWAYS located upstream of the transcribed sequence ON THE SENSE/CODING STRAND. -Wrap our brains around in a 2-dimensional scenario of transcription is that there is no such thing as left and right or right and left in a genome. So genes are just arranged on the top strand on the bottom strand, going left to right or right to left, it's not as easily explained as by this 2 dimensional kind of figure given. so you are not always going to perform transcription left or right sometimes you do it right to left sometimes you will use the top strand as the template sometimes you will use the bottom strand as the template. So in this case we have gene A, gene B, and gene C they are all right next to each other. -Gene A is going to use the bottom strand as the template and is going to transcribe left or right. You know that is because where the green arrow is, is our start site indicating you will do transcription going toward the right. You also know that you were going to use the template strand and your going to read that 3'->5', as you synthesize 5'->3' so the RNA you made from gene A is going to go left to right and is going to use the bottom strand as the templates strand. Gene B though is arranged where the top strand is the template stream and is going to be read right to left. So in gene B the bottom strand is the coding strand and the top strand is the is the template strand. -And then gene C is the same as gene A. Sometimes we also get really complex orientations where we'll actually have gene A,B,&C in the same location and they'll use different segments of the genome within that's location to transcribe their different sequences. No such thing of left to right in the cell. The clues that you're going to use to determine which is the template strand and which is a coding strand is the 5'->3' orientations the location of the plus one and then where the promoter elements are located. -The promoter elements like the TATA box, the CAAT box, the pribnow box in prokaryotes are always located upstream of the plus one and the sequences as we have them written TATAAT the pribnow and TATA sequence, are found on the coding strand. So always the regulatory elements that we care about in the premotor are found in the coding strand which should be at the five prime end upstream on the coding strand which would make the 3' or opposite strand the template strand.

Prokaryotic RNA Polymerase

-RNA polymerase is going to look like, here we have the core polymerase which is made of 4 sub units we get 2 Alpha sub units, 1 beta sub unit, 1 beta prime sub unit, and then 1 Omega sub unit that holds the whole thing together this is what we call the core RNA polymerase. -So this polymerase can actually identify the promoter of the DNA and then it can basically sit down on the promoter but it is not active until the transcription factor called the Sigma factor binds. -Once we bind the Sigma factor to this RNA polymerase core complex this whole thing now to five subunit complex is now called the RNA polymerase holoenzyme and this enzyme is then capable of doing the actual incorporation of RNA nucleotides while it reads the DNA strand.

RNA Structure

-Secondary structure occurs through complementary base pairing on single strand of RNA—makes three dimensional structures (loops and hairpins) -Because RNA is single stranded we can actually get this kind of secondary structures, that are formed through complementary base pairing rRNA and tRNA. mRNA is not really going to do this whole lot but the functional RNA's are going to form these 3 dimensional structures. -rRNA and tRNA are going to form these loop and hairpin motifs, mRNA is going to look more like the primary structure where it's just a long ribbon

Protein-coding genes (mRNA)

-So first is compare and contrast the mRNA or protein coding gene that are produced in prokaryotes and eukaryotes. so on top of this figure it showing you the basic architecture of an mRNA gene for both prokaryotes and eukaryotes. so we always have a promoter it's labeled in yellow, we always have any coding region, and we always have a Terminator. So what the bottom two figures are showing you is a comparison of what the mRNA that's produced from one of these mRNA genes looks like in prokaryotes versus eukaryotes. -on the left hand side the product of transcription for mRNA genes in prokaryotes is the 5' end with an untranslated leader sequence between the 5' end of the mRNA and the first codon that will be used in translation. Then we'll have all of our protein coding sequence, we will have our final codon which in this case is UGA, we will have a 3' untranslated trailer sequence before we reach the 3' end. this molecule is immediately translated so it does not need to undergo any more modifications than this so it is ready to go. the green sequence that we see here which is a protein coding sequence is the exact sequence that is going to end up in the amino acid sequence. -so we'll start with our AUG codon this sequence will be red in the series of three codons until we get to the end, everything in green will get translated everything in blue will not get translated. so the region from AUG to UGA gets translated. those leader and trailer sequences do not get translated that's why they're called untranslated region. There is no 5'cap there is no Poly demolition sequence this mRNA sequence is what immediately starts to undergo translation after it's made. Conversely mRNA that is made in eukaryotes from this gene sequences called pre mRNA and it undergoes a whole bunch of modifications. The first modification is to add a 5' cap to the 5' nucleotide of the 5' leader sequence. -this cap seen in pink here is going to limit degradation of the 5' mRNA molecule. a similar thing occurs at the 3' end or the trailer sequence where we get addition of the Poly identification sequence for PolyA tail this has the same function as the cap on the 5' end. it is going to limit degradation from the 3' end of the mRNA. the third thing we have to do is we have to remove the sequences that are found in the protein coding region that we do not intend to get translated so if you look in the green sequence or the region between the leader in the trailer you'll see a start codon AUG, you will see an end codon UGA, you will see some green sequences labeled as exon's, and you will see some orange sequences labeled as Introns circled in red. the introns are interfering RNA these are sequences that must be removed from the mRNA before this molecule undergoes translation. -So these introns sequences are not meant to code for amino acid sequence in the mature protein, so we need to physically remove these orange intron sequences and splice together the Exon so we have 3 exons on here in the mature mRNA, we want Exon one to be completely ligated to exon 2, which is completely ligated to Exon three we want to remove introns, so the mature mRNA in a eukaryote has to have a 5' cap, a PolyA tail on the 3' end, and we have to remove all of the introns with four the mature mRNA molecule can be shipped off into the cytoplasm for translation

E, P, A sites

-So here is what is E, P, and A sites look like along with a large subunit and small subunit where you can see the mRNA is kind of snaked through these two subunits. So think mostly about like with this bottom panel looks like. what you can see in schematic B here in the bottom left hand side panel is what the ribosome looks like without the mRNA or any of tRNA's without the growing chain of amino acids. Schematic C is showing you with actually going on inside each one of those chambers during the process of translation. -so we have the small subunit we have our mRNA kind of threaded through the middle of the small and large subunits, the E site what it is showing you here is that's an empty tRNA that has already donated the amino acid it brought in it's getting kicked out that's why it's called the exit site. the middle site is called the P site that is where the growing amino acid chain is going to kind of come out of this ribosome so I think of it as like a chimney and then when we make those amino acid, the polypeptide that has to have somewhere to go and so it's gonna shoot straight out the chimney so that's the peptide site. -Then the A site which is on the right hand side here is the site where we bring in the new charged tRNA molecules. so it showing you here is we have our tRNA it's bound to the codon in the A site, so the anticodon an the codon are matched up in anti parallel binding and then what you'll notice is on this tRNA its charged. so it's bringing in the new amino acid up the 3' site and then what we will do is essentially will add that new amino acid to the growing chain of amino acids coming out the chimney in the P site

Transcription Products in Prokaryotes

-So in prokaryotes these are the three types of RNA's that we're going to be producing in prokaryotic transcription will do Messenger RNA, ribosomal RNA, transfer RNA's. All three of these products are used in the process of translation which is the next process will talk about. The mRNA is going to be used as the sequence that you are translating or translating into amino acid sequence. -The ribosomal and tRNA's are going to be useful in that process so the ribosomal RNA's are going to guide the ribosome to the Messenger RNA in the transfer RNA's are going to bring the amino acids to the Messenger RNA so all three of these are going to be needed for translation.

"Charging" the tRNA part two

-So this is an image of what is actually physically happening in the charging of the tRNA so the first thing that's going to happen is we get binding of the tRNA with the appropriate amino acid to their binding sites. so each one binds into the pocket of the tRNA synthetase and then we hydrolyze some ATP to this this process costs energy right we're going to hydrolyze ATP and when we do that the amino acid is going to become covalently linked to the tRNA at the 3' end of the TRNA. -So this is an image of what is actually physically happening in the charging of the tRNA so the first thing that's going to happen is we get binding of the tRNA with the appropriate amino acid to their binding sites. so each one binds into the pocket of the tRNA synthetase and then we hydrolyze some ATP to this this process costs energy right we're going to hydrolyze ATP and when we do that the amino acid is going to become covalently linked to the tRNA at the 3' end of the TRNA.

Elongation

-So this is the process of elongation and it's the same in prokaryotes and eukaryotes. What we're gonna do is start at the 12 o'clock position of this figure and kind of work our way in clockwise direction. So the top figure is essentially what we just saw in the last slide we have our growing amino acid strand centered in the P site, if this is the first step in elongation we will only see one amino acid which will be the initiator methionine amino acid. in this case the growing chain of polypeptide is actually coming out of that chimney. so the first thing that we're gonna do in elongation is we have our new codon that we need to read in the A site. -what we will do is bring in a newly charged appropriate matching tRNA to that A site, the tRNA will bind using its anticodon to the codon situated right in the A site, then we will affectively move to stage 2. so now we found our little tRNA bringing in the new amino acid into A site what we next need to do is form than you covalent bond between the previous amino acid and the new amino acids that we're bringing in. The way we do that is a little bit counter intuitive, what we do first is we hydrolyze the bond so we get rid of the bond between the tRNA located in the P site, and form a new bond between the last amino acid that we added and the new amino acid that we brought in, in the A site. -so we physically chop the linkage between the tRNA and the P site and the growing amino acid chain coming out the chimney, we relocate the chimney a peptide to the A site, then we shift everything down one. so essentially what it showing you in the bottom most 6 o'clock image right is we've shifted the chain over now we have a new covalent bond between the last amino acid we added and the new amino acid. so now the chain is kind of growing out of the A site and then what we do is we just shift everything down to the left right. so we're going to shift the growing chain tRNA which is currently in the A site into the P site, we will shift the new empty tRNA into from the P site into the E site. and then essentially what we've done now is translocated the entire tRNA into the P site and now we have a new open codon located in the A site, we have a growing chain P site tRNA holding onto that chain of peptides that are making coming out of the chimney, we have our empty tRNA in the E site and then we can do is just ejected it. -so then we can move into the 12 o'clock position on this figure again and just start the whole process over again. So the A site it is important for bringing in the new charge tRNA, what we do very quickly early on is we just relocate the growing peptide coming off of the P site onto the A site tRNA then we shift everything down and rinse lather rinse repeat. so we just keep going around in the circle in a clockwise formation until we get to the stop codon. So we bring in the tRNA A site we relocate the chain from the P site tRNA to A site and then we shift everybody down one. also important to note is that you're using two GTP's for every cycle, so every time you relocate the chain from the P site to A site you use a GTP and every time you trans locate the ribosome down one codon you use a GTP

Characteristics of an mRNA gene-- Prokaryotes

-So this is what a mRNA gene looks like in prokaryotes, what you're seeing here in the top part is a double stranded DNA molecule where the top strand is the non-template also known as the coding strand, and the bottom strand is the template strand this is what will be used to make the RNA sequence in the complementary orientation. -The part that's labeled in yellow is the promoter and within that promoter is what we call the upstream region of the gene, so if you look at the line below our little DNA set up, where the little green star is, corresponds to where we're going to start inputting RNA nucleotide. That is what's called the plus one, so that's essentially the transcription start site. So if you go to the left of that transcription start site or on the template strand closer to the three prime end, this is what we call the upstream promoter region -And as you can see if the transcription site is plus one we assign negative values to everything upstream of that so plus one is kind of like your start point everything to the left of plus one in this case is upstream everything to the right of plus one is downstream. So the promoter region is going to be located in the upstream direction of the plus one start site and for every nucleotide upstream of the plus one site we assign a negative number, So we have a -10, -25, -35, and -50. What that implies is that how many nucleotides upstream you are from the plus one orientation site. -So even on here labeled in blue within the promoter at the -10 site is something called a pribnow box. What that means is there's a special sequence located in the promoter 10 nucleotides upstream of the plus one transcription start site, that's going to be essential for starting the process of transcription. So it is a signal sequence inside the promoter that will allow the process of transcription to begin. But the transcription sequence doesn't start incorporating new nucleotides until the plus one, so your plus one is your start site. Everything in purple is the sequence that we are going to transcribe, everything in purple is the sequence that will end up in the RNA molecule that we're making. It will actually transcribe through the end of the purple part and into what we call the Terminator. So the Terminator labeled in red is going to include the stop sequence for those particular type of gene and then termination will end at the end of the Terminator sequence. -So if you look down toward the bottom of the slide the RNA transcript that you were going to make starts at the five prime end at the plus one site, goes all the way through the RNA coding sequences, goes all the way through the Terminator, and then ends at the termination site. So the RNA product that we are going to make does not include the promoter but it does include the termination sequence. so I actually arrange these on the screen such that they are matching where the transcription start sites start and where the transcription is going to end, so the RNA transcript that actually present here starts at the plus one and ends at the terminator. So the promoter although necessary for a signal sequences is not included in the product of M RNA transcription but the Terminator is.

Initiation--Prokaryotes

-Suggest like you saw in the video what we're gonna do now is go through step by step the phases of what happens in initiation, elongation, and termination for the process of translation. so first we'll start with the initiation part of this and so the initiation in prokaryotes is going to be slightly different than the initiation in eukaryotes. so first in prokaryotes we are going to bind the small subunit to the mRNA molecule, and then we're going to do is bind our charged tRNA initiator molecule to the very first AUG which is the start codon. -So even though we have extra nucleotides that are found on the 5' end we're going to start this whole process at the first AUG. so this goes back to when we talked about transcription and modifying the mRNA molecule. the 5' end of this molecule is not the start site so where we are binding are small subunit to the 5' end of the nucleic acid in mRNA that is the 5' UTR. so it is the region between where we started transcription and where will start translation they're different. so the first tRNA comes in and binds a little bit downstream to the start site the first AUG and it has the methionine actually physically located on the tRNA. So this a charged tRNA. The way that tRNA is interacting with the mRNA is through codon anticodon base pairing. -so we all notice here in the zoomed in image and step one is that the initiator tRNA has UAC anticodon and that is base pairing to the first AUG in the codon sequence of the mRNA. AUG is the mRNA sequence that is going to mean methionine. the next thing that happens as we bind the large ribosomal subunit on top of this whole complex and when we do that the tRNA is going to be centered in the P site not in the A site. so this is different from what we'll see in elongation but essentially that first initiator tRNA is going to be centered in the P site not in the A site. Since it is in the P site then when we do that we have to use some GTP so this is an energy requiring step. That is initiation. And what we'll see is when we go into elongation the next codon that will get read is in the A site so we're always reading the next codon on the mRNA in the A site.

Transcription Products in Eukaryotes

-The Product in Eukaryotes are a little bit more complicated, so In addition to those three RNA's we're going to be producing, that we use for the process of translation, we're also going to be producing a number of different types of regulatory RNA's listed in this second group in here. -So will have is the pre-mRNA which is just the precursor to making the Messenger RNA, snRNA, snoRNA, scRNA, piRNA, and what's circled in red here are ones that we're going to talk about more in details

Ribosomal Parts—RNAs have catalytic ability!!

-The large and small subunits made up of a collection rRNA molecules and proteins. In prokaryotes the total ribosome is called the 70S and in eukaryotes we call it the 80S because it's slightly larger so these numbers have to do with how big the molecule looks like when you separate the proteins through protein electrophoresis. •Prokaryotes (total 70S) •Large subunit (50S) •Two rRNAs -23S -5S •Small subunit (30S) •16S -The large subunit in prokaryotes is made up of two rRNA's and one protein. The two rRNA are the 23S and the 5S and that sub unit is called the 50S subunit. the small subunit is made of one protein in one rRNA this is called the 16S rRNA in the whole subunits called the 30S. •Eukaryotes (total 80S) •Large subunit (60S) •Three rRNAs -28S -5S -5.8S •Small subunit (40S) •18S -in eukaryotes the large subunit is made up of 3 rRNA the 28S the 5S and the 5.8S that whole thing makes up the 60S subunit, and then the small subunit is also made up of one small rRNA called the 18S and one protein and that whole thing is collectively referred to as the 40S. it's important to note here that the catalytic capability of the ribosomal subunits has nothing to do with the protein and has everything to do with the rRNA. so the RNA are actually carrying out the catalytic capability of the protein rRNA complexes the proteins themselves are just kind of scaffolding molecules so they are not catalytic the rRNA's are catalytic

Prokaryotic mRNA sequence transcribed from DNA

-The tx start site and the translation start site are NOT THE SAME!!! -It's pretty simple in prokaryotes so the 5' end of the new mRNA transcript that we make corresponds would be +1 nucleotide in the gene so the area that's labeled 5' in blue here the leader sequence that very first nucleotide that's found in that blue sequence is going to correspond to the +1 site in the gene. then we transcribe all the way to the 3' end what you'll notice here is that the translation start site so where we're going to start translating the sequence into amino acid sequence is not located at the 5' terminal end so we always start translation at the codon AUG. -AUG is found further downstream from the 5' end so the region between where the transcript starts and where translation is going to start is called the 5' untranslated sequence or the 5' UTR. so 5' untranslated sequence means that this region between the start of the mRNA and the first codon is not going to be included when we do translation into amino acids. we will start translation at the first AUG which is the first nucleotide in green in this picture. we will end translation at the stop codon which is the last nucleotide labeled in green of this protein coding sequence. -You will also notice is that the stop codon is not the end of the sequence so we also have a 3' untranslated sequence at the 3' end so it is not included, we have additional mRNA sequence on either side of the protein coding sequence and the reason for this is that we have to collect all of the enzymes that are going to start the process of translation In an area and we have to have a way for them to have a termination. so thinking ahead on either side of the protein coding sequence such that the transcription start and end sites are different from the translation start and end sites

Eukaryotic protein-coding genes Termination and synthesis of the poly(A) tail

-This is going to occur during termination so this is a close-up you seen this figure before what is happening during the process of termination. so at the top you see our pre-mRNA molecule, we have our Poly a signal which is AAUAAA that is going to bind to a series of proteins which will hold the molecule steady while it's being trimmed, so it's going to get cut at the cleavage site and then what it's showing you in this little red circle that's labeled with a cut is that the area that is attached to the RNA polymerase in DNA molecule that continuing to be transcribed, that used to be attached to the 3' region of the mRNA molecule that was being produced. -what we've done here is we've trimmed that so we've cut the molecule that's being produced by that RNA polymerase off at the cleavage site and then as soon as we perform that cleavage where do we add the PollyA polymerase chain of adenine nucleotides directly to that 3' end and then that's the additional 3' polyA tail. the RNA polymerase will continue to transcribe useless sequence from the DNA and then rat1 is going to bind to where that cut site was on the RNA that's still being produced and it will chase the RNA polymerase and degrade that garbage molecule as it continues. but our pre mRNA that has our new Poly a tail attached will now be protected from the 3' end and just like it's protected at the 5' end

Addition of polyA tail Post-transcriptional During termination Addition of Poly Adenylation Sequence

-This is our pre-mRNA that we have made from transcription. we have our consensus sequence and we have our cleavage site that is going to be recognized by our termination enzymes. So what will do is will leave at the cleavage site producing this little 3' end and once we've produced that 3' end of the pre-mRNA we're going to add a Polyadenylation sequence that has been manufactured by a special polymerase called polyA polymerase. -so this tail is approximately 250 nucleotides long they're all the same nucleotide adenine and this gets added to the 3' end of our mRNA, so that we basically provide a little bit of buffer between the end of the sequence that is going to be translated and the end of the molecule. so we still have our 3' end but now if this molecule becomes degraded from the 3' end and we had to get through 250 nucleotides before we start eating into the sequence that we're trying to translate. -so you can think about this Poly a tail very similarly like a telomere in DNA so it's basically we've added a bunch of buffer sequence to the end such that if we start to degrade this molecule from the 3' and we have to get through a bunch of garbage sequence before we start degrading the sequence that's going to get translated

Final tRNA After Processing

-What it is showing you on the left hand side is what the actual space filling model of the tRNA looks like so it almost looks like this little R shape and we have a couple of different domains here. So in the second panel what is showing you is how the hydrogen bonds are holding the secondary structure together where the 5' end is versus where the 3' end amino acid attachment site is and then in the bottom of this R shape showing you the entire codon arm. -So labeled in purple is the anticodon, that is the part that is going to interact with the mRNA during translation to read the codon sequence in the mRNA. So what it showing you in the last panel is the kind of cartoon version of how we draw a tRNA and the three major domains that we have. so we have the DHU arm we have the T5C arm and then we have the anticodon arm which is really the only one that you care about. this is the part that's going to be necessary for our translation capabilities, so tRNA's are going to physically bring in the amino acids during translation and read the mRNA with this anticodon arm. -every place you see a red dot is a nucleotide that was modified so these are what we call rare bases or bases that are chemically modified so that they're not the typical ACG or U. Then as you can see the CAA is in Gray that's added up the amino acid 3' attachment site, so tRNA's are heavily modified and the reason they're heavily modified is so that they can perform their functional purpose in translation

Splicing and Editing of tRNA

-here is the general process that occurs in the editing of tRNA's we do call this splicing but it is a completely different process and then the splicing in mRNA so just making note of that, it's not splicing of intron's really it's just splicing of extra material in the tRNA. The reason they're not true introns is because we don't do true alternative splicing of tRNA so even though in this figure it's labeled as intron is not really an intron because the intron terminology implies that that your going to do alternative splicing, which does not occur in tRNA's. -So the first thing we are going to do in step one is that we have all this extra material that's produced in this tRNA, and it's showing you the tRNA in its secondary structure. So tRNA is our functional RNA's that do get folded into kind of like this hairpin formation that has a couple of different domains. Essentially anything that's located in this kind of light blue green color needs to get removed so we have our 3' and we have our 5' end they both need to get trimmed and then we have this kind of a large intron in what will form the anti-codon arm that also has to get cut out. so the precursor RNA is made and then what we need to do is trim the 3' end and 5' end, so that's the first thing to go we actually get rid of the terminal extra RN. -And then the second thing we do is we trim out this quote intron at the anti-codon arm. So same process occurs we do the 5' splice site, we loop it to the branch point, we cut the 3' splice site and then what ends up happening is we move that little anticodon region that it's showing you into the bottom terminus of this stem loop. So we need that little anticodon to be right at the bottom of that loop formation. Then what we do in step three is we add the same three bases to the 3' end so we actually not only get rid of excess RNA here we actually add some nucleotides to the 3' end. we always add CCA or ACC in the backwards orientation to the 3' end. -The reason we do this is that at the end that is going to get physically attached to the amino acid during translation. And so all of the tRNA's have to have that exact sequence at that exact site such that we can add the amino acid to the 3' site. so then once we do that we actually physically go back and chemically modify some of the nucleotides in step 4. So we modify the bases such that this tRNA is now completely functional. so every place with a red dot in panel 4 is a nucleotide that gets chemically modified so that the tRNA is completely functional

Complex of transcription factors + RNA Polymerase II

-so here is showing you an example of why we might use an upstream regulatory promoter element. Essentially what is in yellow is the core promoter and then it shows you the plus one transcription start site kind of at the Terminus of that core promoter. -In orange is what we call an upstream activator element or regulatory promoter and what we can see here is that we're going to collect a whole bunch of proteins on the core promoter itself including the RNA polymerase, this will get us what we called basil transcription which means that will get some transcription from this gene but not a high level of transcription for this gene. -So if a eukaryote wanted to crank up transcription from this particular gene would it could do is basically bend the DNA around using that regulatory premotor and what's called a upstream activator sequence, and through the coordination with enhancer proteins and activator proteins it will basically bend the DNA around. -Such that enhancer proteins and activator proteins can kind of stabilize this whole complex through the activation of a mediator. So basically folds the DNA over and then it makes it much easier for the cell to coordinate all of these proteins binding to the promoter, and the outcome of that is that we will get much higher transcriptions that if we were not bending this DNA and stabilizing that protein DNA interaction. -So this is kind of the general rule of a regulatory promoter is activation of transcription but we can also get what's called silencing regulatory promoter that will destabilize or have the opposite effect by bending this DNA around. So these are additional activities that eukaryotes can carry out to fine tune their RNA transcript function of specific gene this doesn't occur all the time, its gene specific, and this does not occur prokaryotes only occurs in Eukaryotes.

DNA template strand-> mRNA-> Protein

-so this is showing you going from double stranded DNA sequence we're going to transcribe one of those sequences using the complementary base sure our name you're going to modify this heavily go ahead and use the mRNA in either case we are going to use the sequence in the mRNA molecule to undergo a translation step that is going to get us out of nucleic acid language into amino active language -so in this image what is showing you is the way that we do that is by entering the ribosome over three nucleic acid molecules at a time so three nucleotides will form a triplet codon and that triplet codon will form the basis of the translation from the nucleic acid language into amino acid language in this case it showing UGG is the first codon followed by UUU the second codon, GGC is the third codon, UCA is the 4th codon. -In each one of those triplet codons is a translated into an amino acid language so we're going from nucleic acid language which is red and three nucleotides at a time and that is getting translated into amino acid language so UGG codes for the amino acid tryptophan

The Wobble Base of tRNA

-so this is what makes redundancy in the universal code possible. so we have a codon in the mRNA this case it's UCC. UCC means serine but also UCU can mean serine, so the reason that we have some redundancy in the universal code meaning that multiple codons can code for the same amino acid is what's called the wobble base phenomenon. so in a tRNA molecule really the only two parts of the codon that matter are the first 2, so the 5' most nucleotide and the middle most nucleotide. -The third nucleotide in a codon is often referred to as the wobble base or the wobble position and that is because it's not really necessary in the reading of the codons. so AGG is the anticodon, that matches UCC so that will be found on the tRNA molecule but AGG even though it's not a perfect fit to UCU because those first 2 nucleotides are a perfect match we're still going to bring in serine using this tRNA molecule in the anticodon. so that's what's called the wobble position the 3' end does not need to be a perfect match

tRNA Processing

-tRNA genes not found within rRNA transcripts but are produced as pre-tRNAs-need to be modified -Alright so just as a reminder prokaryotes tend to group their tRNA's into rRNA gene transcripts. eukaryotes don't do that they produce their tRNA is all by themselves from their own genes, but essentially what we make from this process specifically in eukaryotes is that we make a pre-tRNA molecule that then has to be trimmed and modified similarly to the process that occurs in mRNA

Subunits look like

-what those different subunits look like. so in the prokaryotic periodic subunit you can see the large subunit has two rRNA. the small subunit has one rRNA and then collectively together that whole thing is referred to the 70S. Then in eukaryotes have three rRNA's in the big subunit one rRNA small subunit and then collectively that whole thing is the 80S but you can see how they have homologous features

mRNA sequence: 5'-AUGUUUAAAAGUCUGUGA-3' 5'-AUGUUUAAAAGUCUGUGA-3'

5'-Met-Phe-Lys-Ser-Leu-STOP-3' -** You will be responsible for knowing what each 3-letter abbreviation means (Full name of amino acid) -So here is your universal codon chart the codon chart is always read in mRNA language so you are always looking at the mRNA sequence and then trying to determine what the codon pattern is for translating this into amino acid .so for example here is the mRNA sequence located at the top left hand side of the screen AUG is always the first codon red so the translation start site in our mRNA sequence is always going to be the first 5' AUG sequence this is also very important because it sets up what we call the universal code reading frame so anytime you're trying to translate in a sequence into amino acid sequence and you scan down locate the first AUG and that is your first codon. -So what I've done here into the sequence of interest is I've highlighted the first AUG and then grouped these nucleotides into units of three based on that first code. In this case AUG always means the same thing it means methionine and it means start so it means that that is the first we're going to incorporate in our knew amino acid sequence that we're making so start. UGA the special one it's one of three stop codons so these are one of the three fancy triplet codon sequences that does not code for a an amino acid it just means that you're going to terminate translation so UAG,UAA, and UGA all mean stop so in this case you incorporated 5 amino acids and then the sixth codon doesn't incorporated amino acid it just means stop

mRNA sequence: 5'-AUGUUUAAAAGUCUGUGA-3' 5'-AUGUUUAAAAGUCUGUGA-3'

5'-Met-Phe-Lys-Ser-Leu-STOP-3' -** You will be responsible for knowing what each 3-letter abbreviation means (Full name of amino acid) -We will talk about the redundancy that's built into the mRNA universal code. So what you might notice when you look at this as there are several codon sequences that that code for the same amino acid in mRNA. so like for instance CCU, CCC, CCA, CCG those all code for the amino acid proline and what you might notice is that the only way that these codons differ is by their 3' nucleotide. so CC X meaning CC whatever the 3rd nucleotide is in the codon, they all mean the same amino acid

Termination Sequences and the Rho protein

Bacteria: Rho-dependent termination -Rho dependent termination the poly uracil sequence is not enough to make determination spontaneous. So what we need is an additional protein called Rho that will chase down the RNA polymerase and physically unwind RNA polymerase from the DNA and the RNA from the DNA. So the same general series of events happens we have our transcripts being made, we continue to transcribe through those anti parallel complementary sequences so that we formed the hairpin. -When we form the hairpin the RNA polymerase pauses just like it does in Rho independent termination, but what happens in Rho dependent termination is that the polymerase will just keep going, so it will stall at the Poly uracil sequence but that does not stop transcription, it stalls out and then it keeps going. So what we need here because the polymerase basically doesn't obey the sequences in the spontaneous method of termination is we need an additional protein called Rho to basically force the RNA polymerase to stop transcribing. -So the Rho protein binds to the mRNA closer to the 5' end and it chases the RNA polymerase until it comes in contact with the hairpin. When the RNA polymerase stalls briefly at the hairpin it gives the Rho protein a chance to catch, so it basically get hung up on the hairpin the Rho protein catches up to the RNA polymerase, and then once that happens the Rho protein physically causes a dissociation between the RNA polymerase, the DNA, and RNA. So it causes this whole complex to fall apart.

Termination Sequences

Bacteria: Rho-independent termination No proteins needed -so this is what is happening in the Rho independent version of termination so as we're making our little green RNA molecule we will transcribe through 2 hairpin sequences. So what it showing in these highlighted in these orange sequences are anti parallel complementary sequences that will help us form hairpin with the mRNA molecule that we are synthesizing. -So as we are moving the RNA polymerase down towards the right hand side we will first make this original sequence a AGCCCGCC going from 5'->3'and then we will continue to transcribe, we'll get to another one of those sequences which is the anti-parallel complement to the first sequence and then we will hit what's called the Poly uracil sequence, which is just right after this last orange sequence. What will happen is the RNA as it's being transcribed will form this complementary base pairing will form a secondary hairpin structure right before we hit the polyuracil sequence. -When that happens, it causes the RNA polymerase to kind of stall for a second, so RNA polymerase is elongating incorporating new nucleotides it hits this hairpin and then it essentially like stops just for a second it hesitates. Then it starts to incorporate the multiple uracil's in the polyuracil sequence, which is just encoded by a bunch of adenines on the DNA sequence, and when it does this the A and U base pairing between the RNA and DNA destabilizes the interaction between the RNA molecule the DNA template and the RNA polymerase. -When it does that the whole complex just falls apart so the RNA polymerase falls off the DNA, the RNA falls off the DNA, the two strands of DNA snap back together and then essentially we have our three components returned to their original state. We have our double stranded DNA, our RNA polymerase is now free to transcribe another gene, and we have our mRNA molecule with little hairpin at the end. -So the reason that this happens or the reason the Poly uracil sequence destabilizes this whole complex is because there are only two hydrogen bond that take place between adenine and uracil. We have a stretch of multiple Adenine and Uracil bases it weakens the interaction between the RNA in the DNA because we don't have any of the GC bases to kind of strength in the interaction between the DNA and RNA, so really independent termination we form the hairpin and then we have the poly uracil sequence and this pretty much just causes the complex to spontaneously dissociate.

Characteristics of genes coding for rRNA-Eukaryotes

ITS-internal transcribed spacer ETS-external transcribed spacer ** Both present in rRNA transcript; both removed during post tx modifications* -Eukaryotes don't do that exactly so in eukaryotes we tend to incorporate promoters within the transcribed spacers. So in this case we had a promoter that's found in the external transcribed spacer and then we repeat this unit of ribosomal DNA so we'll have multiple of these units sequence and so the promoter will start in the first left hand side ETS will make the 18S rRNA the 5.8S rRNA and then the 28S rRNA gene altogether before we hit the Terminator -and will chop them apart in those spacer regions once we get our full transcript. so this is something that does not occur in eukaryotes mRNA genes in mRNA genes we always have one promoter, 1 gene sequence, one Terminator at the end. in our RNA genes we have multiple genes that are under regulation of a single promoter and a single Terminator

Polycistronic mRNA transcript (Prokaryotes only)

Some prokaryotic mRNA may be polycistronic Example: lac operon -So even though mRNA that's produced in prokaryotes does not undergo the immense amount of processing to convert pre-mRNA to mature mRNA just like it does in eukaryotes we still do some limited processing in the prokaryotic cell of mRNA. So the reason that we do this is because some prokaryotic mRNA is produced as a Polycistronic. -So what this term polycistronic means is that the genes that are regulated in prokaryotes are often arranged in sets so there are range in what is called an operon. What you're seeing on the figure is the most classic example of an operon gene arrangement in prokaryotes. Whereby we have one promoter and then arranged behind that promoter we have three independent genes before we get to a Terminator so we have one promoter, multiple genes, and a single Terminator. this is called polycistronic operon so as this mRNA is made it is made as one long transcript celled the lac mRNA transcript in this case, but the transcript consists of three genes. -So what's going to happen in prokaryotes is this one mRNA gets made and then the transcript is going to get translated and when it gets translated we will produce three independent proteins from that single transcript. so sometimes the mRNA will get chopped into three pieces other times the mRNA will get translated into one long peptide and then the peptide will get 3 pieces. what's different about this gene arrangement than eukaryotic gene arrangement is that eukaryotic gene arrangement is always single promoter, single gene, single Terminator for mRNA. -prokaryotes do one promoter several genes one Terminator the reason that they arrange their genes into operons or into Polycistrons is because all of these genes will be associated with one single pathway so if they want to turn on a specific pathway all they have to do is turn on one promoter that guarantees they get expression of all three genes that are related in function together and then essentially this allows them to turn on certain functions in turn off certain functions all in one go. -so they don't have to worry about turning on gene A and a pathway, gene b, and then gene C all independently they turn them on all together they produce this long mRNA transcript and then all the genes that they need for a specific function are being produced at the same time. eukaryotes have a completely different way of organizing their genome

Elongation of Transcription - Synthesis of the RNA Polymer

Template (antisense/non coding/template strand) = read 3' to 5'Strand complementary to template (sense/coding strand) = 5' to 3' Direction of new polymer synthesis? -So elongation of transcription Is basically the synthesis of the new RNA primer. What is showing you in the figure is you have opened up a transcription bubble and the RNA molecules that's being synthesized in the 5'->3' direction where your building new RNA nucleotides on the free 3 prime OH of the preexisting nucleotide. -And then we are using the template strand as a way to inform which type of nucleotide we are going to be inserting, the sequence that we generate is going to be anti-parallel and complementary to the template strand but is going to be identical to the non-template or coding strand. The only difference here is that were we see Thymine in the non-template or coding strand we are going to put in uracil. -So in this example what you're seeing is the template strand sequence 3'->5' is ATGCCTATGC, the knew RNA molecule that we make is made in the anti-parallel faction so it's made 5'->3' and now we're going to put in the compliment, so we put in UACGGAUA and then eventually we will put in the C and G. This sequence of new RNA polymer that we have made matches the coding strand so if you look at the strand that is not being used by the RNA polymerase as a template it matches the RNA, the only difference here is that the uracil is now standing in the place of thymine.

"Charging" the tRNA

This process provides specificity of translation: •Each amino acid uses a single tRNA synthetase •Enzyme is specific for BOTH the amino acid and the tRNA •Only 45 tRNAs for 61 codons—redundancy! -the process of translation just kind of occurs all the time in the cell is not really part of the process of translation just kind of always happening but it's necessary to facilitate the process of translation is what's called charging the tRNA. so there's a very fancy group of enzymes called tRNA synthetases and they are responsible for matching up the tRNA molecules with the correct or appropriate amino acid so what it is showing in the figure is that you have the tRNA synthetase that is specific for the amino acids and tRNA's that are going to carry the specific amino acid tyrosine. -so for each tRNA molecule we're going to have a slightly different enzyme but they're all under the large umbrella classified as tRNA synthetases. Each tRNA synthetase has 2 binding pockets and has a pocket for the tRNA that matches the anticodon sequence that goes with the codon sequence of the mRNA sequence for in this case tyrosine. so the anticodon is gonna be AUA which means that anticodon is going to read the codon sequence UAU which matches up to tyrosine in the mRNA molecule. the second binding pocket is for the appropriate amino acid that will match the codon in the mRNA so tyrosine is going to match the codon UAU in the. -so this enzyme is responsible for matching up the appropriate amino acid with the tRNA that will match up to the codon in the mRNA. so each amino acid is going to use a different tRNA synthetase, the enzyme is specific for both the amino acid in the tRNA. we have 45 tRNA's for 61 codons which is accounted for in the redundancy of the universal codon table. what does enzyme is physically going to do is link covalently that tyrosine or that amino acid to the tRNA and then when we go to do the process of translation. The tRNA will bind to the mRNA molecule in the ribosome and it will bring in the amino acid that matches the codon

Addition of 5' cap Co-transcriptional Cap Structure at the 5¢ End of a Eukaryotic mRNA

Typical 5'->3' phosphodiester bond is subject to degradation with exonucleases Addition of guanine in a 5'->5' phosphate linkage is not subject to endonuclease degradation Cap protects mRNA from degradation (at the 5' end) -The addition of the 5' cap happens during elongation. so as soon as the 5' most nucleotide exits the RNA polymerase as with little kind of RNA is released out of the RNA polymerase we get some enzymes that bind to the 5' end of this new RNA transcript, and they're going to attach a guanine nucleotide to the phosphate group of the 5' nucleic acid. so the way that we attach one of these one in caps is not through the traditional 3' to 5' linkage that we see where we generate phosphodiester bond we instead flip the nucleotide around and attach the 5' carbon to the 5' carbon of the pre existing 5' nucleotide. -So instead of a 5'->3' linkage which would create a phosphodiester bond we add a 5'->5' linkage which is not a phosphodiester bond which means the linkage that we create is not subject to degradation by endonucleases or exonucleases. So what is showing you on the left hand side of this figure was highlighted in orange is your guanine nucleotide that's getting attached this is your 5' cap. and what is happening is the 5' carbon is going to get attached to the 3rd phosphate group on the first 5' nucleotide of your growing RNA molecule. -so this is the 5' to 5' linkage which protects the 5' end from degradation, because there aren't any enzymes that recognize this 5' to 5' linkage. so there are no enzymes that can degrade the 5' end now so when you add this 5'cap onto this nucleic acid. essentially what it does is it makes this region of mRNA invisible to any of the endonucleases that are floating around in the cell. so essentially it's going to protect the 5' end of this mRNA from degradation

What are the Features of the Genetic Code?

•4 nucleotides arranged in groups of 3 •Yields 64 possible triplet codons (43=64) •Only 61 codons code for amino acids •3 stop codons—no amino acids correspond to these codons •Redundancy—Several codons can mean same amino acid •No ambiguity—Each codon only ever corresponds to one amino acid •Universal code (applications?)—ALL organisms use this system!! •Continuous reading frame—no skipping nucleotides •UUU broke the code!! -features of the genetic code so this is basically how we're going to translate the nucleic acid language into amino acid language which will ultimately form the building blocks of the protein. So we have four nucleotides that are contained in mRNA molecule so we have AGC and U and we arrange them in groups of three so four nucleotides arranged in groups of three yield 64 possible triplet codons but instead of using all 64 possible codons. -universal codon only uses 60 one of those codons to code for amino acids the other three codons code for the stop sequence so 4 three of the codons that I'll show you in the codon table of those 64 codons three of them don't code for amino acid code for determination sequence in Process of translation. There is also a large amount of redundancy in the genetic code so several codons code for the same amino acid there are only 20 essential amino acids so of those 61 codons that code for amino acid only 20 amino acids will get input so there is a large amount of redundancy so I think there are six codons the code for a single amino acid, most of them coded for by 4 different codons. -there is no ambiguity in the genetic code meaning Each codon only ever corresponds to a single amino acid so even though there might be 4 codons that mean the same amino acid, it is always those same 4 codons that mean that amino acid. There's no ambiguity so there is no wiggle room in the universal code. the code is universal which means that all organisms use the exact same system so a bacteria, a archaea, Eukaryote they all use exactly the same universal code and we'll talk about the meaningful applications of this later on Continuous meaning that it is read in three nucleotide groupings back to back so there are no skipping nucleotides it's arranged in nucleotides 1, 2, and three. that means one codon and then the next codon is immediately a budding that codons at one two and three is codon number 1. 4,5, and six is codon number 2. -There is no buffer sequence between codons and then the way that scientists discovered this unique universal code or how the translation machinery recognized the nucleic acid sequence was by using a long string of mRNA that was just a bunch of uracil's so you broke the code

Universal Code

•ALL organisms use the same genetic code to translate mRNA to protein •Important application in generating transgenic organisms •Ex. Insulin (Diabetes used to be lethal) •Treatment for diabetes •Used to harvest insulin protein from pigs •Now, we used bacteria to produce human insulin BECAUSE the codons are universal •** Evidence of evolution -So the universal code is just that it's universal meaning all organisms use the same genetic code to translate mRNA into protein and this is very important because we as geneticists are able to create transgenic organisms meaning organisms that contain the gene from a different species. and we can create an example the protein insulin for humans in a completely different organism. so we are actually able to genetically engineer one specific organism to produce the proteins from a different Organism because the universal code is the same. -so essentially in this example you can take a gene from a human which is the gene for insulin you can insert it into a plasmid and stick it into a bacterial like E. coli. then the E.coli will just produce the human insulin the reason for that is that the universal code is interpreted the exact same way in every different organismal species so this is actually evidence of evolution Because basically all organisms on planet earth use exact same universal code

Initiation Complex Activation

•Activation of pre-initiation complex through binding of other factors 1. RNAPol II phosphorylated= ACTIVATION! -In order to activate this complex and make the open complex, such that this can start incorporating new RNA nucleotides and start reading the template strand of DNA, we have to activate this complex. So to go from the pre initiation to the open initiation complex we have to phosphorylate the RNA polymerase. -So once these complex forms, another enzyme will come in and phosphorylate the RNA polymerase (well activate it) and then we form it's called the open complex I then we move into elongation.

tRNA

•Carries amino acid to the ribosome complex •"Reads" mRNA codon with anticodon -So first we're gonna do is discuss the tRNA and you've seen these before we talked about acid structure but essentially these are functional RNA so these are RNA molecules that do not undergo the process of translation but they're used in the process of translation just as they are/ so they are RNA's that are going to get folded into 3 dimensional structure they are basically going to be the unit in this whole process that are responsible for performing the translation. -So therefore there is this 3 dimensional structure and the two components that you really need to focus on for these tRNA's are the 3' end which is always modified to have CCA going from 5'->3' at the 3' and that is the part that is going to physically get attached to the amino acid. and then the second component is the anticodon arm so that's located at the bottom of the tRNA this is going to be a three nucleotide component that is going to be responsible for binding to the codon Of the mRNA in an anti parallel complementary fashion and that is what is going to physically determine if that tRNA molecule is bringing in the correct amino acid. -so we'll look at what that looks like in the next series of slides but essentially the tRNA is the molecule that does the translation, so it reads the codon in the mRNA with its anti codon arm and physically brings in the correct amino acid to the matching mRNA codon sequence. Draw your attention to in this slide is that we're not going to draw the tRNA's in their three dimensional form we're going to draw them kind of cartoons so the third panel in this in this figure shows you this kind of little like upside down stocking. So there's like a little region where the toes would go that's the amino acid start site that's our 3' end and then on the bottom side it shows you the anticodon so in this case from 3'->5' front you have AAG that is going to bind to the codon sequence in the mRNA

Differences in RNA Processing

•Colinearity of gene expression occurs in prokaryotes only •Couples transcription and translation •Eukaryotes produce pre-mRNA •RNA is processed in nucleus BEFORE export to cytoplasm for translation -So first we need to do is talk about the differences in RNA processing in prokaryotic cells versus eukaryotic cells. so the reason we need to distinguish between these two cell types is that periodic cells have a barrier between where transcription occurs and where translation will occur this is all the nuclear envelope and thus the system of gene expression or producing protein is going to have a natural pause between when we perform transcription we perform translation. -So essentially what is going to happen is this additional step that occurs in the process of transcription and translation where we produce pre-mRNA from the process of transcription within the nucleus and then the is going to undergo a whole bunch of modifications while it's still in the nucleus before it gets shipped out into the cytoplasm to undergo the process of translation, so there's functionally a barrier that separates the two processes. -in prokaryotes though there is no barrier because there is no nuclear envelope so the RNA that is being produced in process of Transcription is immediately translated as it's produced so as that mRNA is being produced from the DNA template we're actually going to get immediate translation of that mRNA transcript. so there is no way to stop and pause and modify the mRNA that is getting made from the DNA moleculee before it starts to get translated, this process is called Co linearity of gene expression and it only occurs in prokaryotes because there is no nuclear membrane that is going to separate these two processes of transcription and translation. -Eukaryotes cannot do that so there is no collinearity of expression in eukaryotic cells. And the mRNA that is going to be produced in eukaryotes will be referred to as pre-mRNA and then it will undergo processing before it gets shipped out and undergoes the process of translation

Module 7 topic 2 Topic Outline

•Compare necessity of processing in eukaryotes to colinear expression in prokaryotes •Processing events in eukaryotic premRNA->mRNA •Processing events in rRNA and tRNA •Produced as one RNA with several genes; genes get cut into individual units •tRNAs are adjusted (trimmed) •Regulatory RNAs •Processing •Function

Initiation & Elongation

•Core polymerase and sigma factor bind to promoter •Double stranded DNA is unzipped—produces a transcription bubble •Template strand is used by polymerase to incorporate complementary RNA polynucleotide 5'->3' •Same kind of process as DNA replication •Unidirectional!! -After we put our core polymerase onto the promoter it basically just sits there, as the core RNA polymerase, until a Sigma factor is freed up to then bind to the core polymerase. so that's what showing you in the figure on the right so in panel A the core polymerase is binding to the promoter it's recognized that is a gene that it may be wants to transcribe, but it will not transcribe the gene until sigma factor joins the party. -So then the sigma factor, when we get available sigma factor, will bind to the core polymerase making this whole complex now called the holoenzyme. And then when we bind that sigma factor we basically activate the RNA polymerase capability of the complex. So the first thing that happens is we unzip the two double stranded DNA molecules from each other so we create this little bubble ,so just like in replication what we need is to start transcription is a single strand of DNA. -So we want to separate those two strands of DNA away from each other, so we do this very similar thing like we do in DNA replication where we unzip this kind of double stranded DNA molecule. When we do that we worn up called a transcription bubble in this case the bubble is actually found within the enzyme complex, so it's not exposed to the outside area the bubble is actually found on the inside of the polymerase so that's what it's showing you in panel C. -And then what's going to happen is once we unzip these two strands of DNA from each other we're going to use the template strand in this case the bottom strand as a way to incorporate nucleotides in a complementary fashion when we make our new mRNA molecule. As we do that we will incorporate new nucleoside triphosphate, as this RNA polymerase complex slides on down to the right. So essentially this will keep sliding and as it slides it will open up that bubble and behind it, the bubble will close back together so essentially we have single stranded DNA within this RNA polymerase and then as he RNA polymerase moves to the right, It unzips the DNA down to the right and then the DNA gets it back together following enzyme movement. -So the only place where we're having single stranded DNA explode is inside this RNA polymerase complex. The process of making new mRNA is exactly the same as making a new molecule of polymeric DNA, we are going to incorporate new nucleoside triphosphate and we are using DNA in this case as our template to make the RNA molecule. So what is showing you in panels D and E is the incorporation of the new nucleotides as we slide that molecule down and the sequence that we are going to generate will be complementary to the template strand made in a 5 prime to three prime orientation. -So remember you can only build on to three prime OH as you run down, so the generation of the new polynucleotide follows all the same rules as in DNA replication so we still synthesize from 5 prime to 3 prime but now the sequence is the exact sequence found in the coding strand which is the strand that is not being used as the template the only difference is where you see a T in the DNA sequence you are now going to incorporate uracil instead

Learning Objectives

•Describe the process of transcription (including stages) •Prokaryotes vs. Eukaryotes •Enzymatic machinery and DNA elements used in tx •Product(s) of transcription •Describe the elements of the transcript present after transcription, that are destined to be removed during RNA processing** •Organization of a gene •Elements that control transcription •Differences in gene arrangements •mRNA (prokaryotes vs eukaryotes) •rRNA •tRNA •Describe the advantages of colinearity of expression in prokaryotes and explain why this does not occur in eukaryotes

Module 8 Topic 1 Objectives

•Describe the qualities of the universal code •Use the universal code to translate mRNA into protein •Describe the process of translation of mRNA for both prokaryotes and eukaryotes •Machinery & components of the ribosome complex •Sequence of events—be able to draw the process of elongation in the ribosome complex (A, P, E sites) •Use of triphosphate nucleotides (GTP and ATP) •Utility of the wobble base •Describe the major types of point mutations and their consequences •Be able to determine the type of mutation if given an example

Types of Genes Classified by RNA product of transcription

•Genes in both Prokaryotes and Eukaryotes •Protein-coding genes (transcribed into mRNA) •Ribosomal RNA genes aka rDNA (transcribed into rRNA) •Transfer RNA genes aka tDNA (transcribed into tRNA) -The types of gene are going to be classified by RNA product of transcription. Here are the genes that are present in both prokaryotes and eukaryotes mRNA, rRNA, tRNA those are common to both groups. •Genes only found in Eukaryotes •Pre-mRNA (product of mRNA transcription BEFORE processing) •Small interfering RNA (siRNA) •MicroRNA (miRNA) -Then there are genes that are unique to eukaryotes those are going to be pre M RNA which is a specific type of RNA that is produced in the nucleus before it undergoes processing to become the mature mRNA. The reason we say that this type is unique to eukaryotes is because like we said before prokaryotes do not pause between the process of transcription and translation. -So there is no time in prokaryotes to produce an immature version of mRNA because processing does not in prokaryotes. But it does occur in eukaryotes so before we produce are mature mRNA the molecule that we produce is first called pre-mRNA and this is what it's called before it undergoes processing in the nucleus it gets shipped out on to the cytoplasm. -The other two types of RNA genes that you need to care about that are specific to eukaryotes are SI small interfering also known as silencing siRNA and micro miRNA, these are two types of regulatory RNA that will talk about.

Phosphorylation Forms Open Complex

•Phosphorylation of RNA Pol II opens DNA to expose template strand •Phosphorylates RNA Pol tail to induce activation •When elongation begins, RNA transcript is formed using the complementary strand, not the coding strand! •Generates RNA •Transcript from 5'->3' •(reads template strand 3'->5') -The diagram shows what's going on inside that RNA polymerase complex. So once we form the open complex we essentially unzip the two strands of DNA, so we disrupt all of those hydrogen bombs that are found holding the two DNA molecules together, the way that we do this is basically RNA polymerase has this little spool in the middle where it kind of gets wedged between the two DNA molecules forcibly separating it as this complex slides down to the right. -So we phosphorylate , we unzip the DNA or unspool the DNA, and the as we move into elongation this whole complex is going to move down it's going to read the template strand in the 3'->5' orientation. While it does that it's going to synthesize new RNA Poly nucleotides that are complementary to the base pairs that are being read on the template strand or identical to the base pairs that are not being read on the coding strand. -So we're going to generate new RNA you're going to make or synthesize this RNA transcript in the same direction that we've been doing thus far 5'->3', but when we do that because DNA and RNA are anti parallel binding molecules we're going to read the template in a 3'->5' prime orientation while we generate the new transcript in the 5'->3' orientation.

Mutations in the DNA sequence

•Point mutations—1-3 base pair sequence changes in the DNA sequence •Leads to RNA sequence change •Potentially changes the amino acid sequence in the protein produced •May alter the function of the protein produced •Types of point mutations: -Silent -Missense -Nonsense -Frameshift -Point mutations in the DNA sequence, and point mutations are list of mutations so they are essentially between one and three base pair sequences that are altered in the DNA. That DNA sequence change leads to a change in the RNA sequence and that can potentially lead to a change in the amino acid sequence which may or may not change the function of the protein that's produced

What new things did we learn about mRNA transcription?

•Product - different types of RNA •Molecular toolbox for transcription •Gene sequences •Types of gene structures •Types of DNA signal sequences •Types of RNA polymerases •Specificity of initiation and termination •Prokaryotes and eukaryote differences

**Pay Special Attention to the Following**

•Product - different types of RNA produced by transcription -you're going to pay attention to the product of RNA that you're making so this could be mRNA, rRNA, tRNA or those regulatory types of RNA. •Molecular toolbox for transcription •Types of genes: organization (structure) and DNA signal sequences •Types of RNA polymerases •Specificity of initiation and termination •Prokaryote and eukaryote differences

Summary of RNA Processing

•Prokaryotes •Produce mRNA; rRNA; tRNA •Process rRNA and tRNA •mRNA is frequently polycistronic genes cut apart AFTER transcription -Prokaryotes produce mRNA,rRNA, tRNA they're going to specifically process rRNA and tRNA through methylations but they process mRNA in a very different way than we process eukaryotic mRNA. essentially all we're going to do for the processing of mRNA is chop individual genes apart from one another so they don't undergo capping or tailing they don't undergo intron splicing or alternative splicing we physically or just gonna cut the genes apart from one another. •Eukaryotes •Produce pre-mRNA; rRNA; tRNA; miRNA; siRNA •Process all of them (different methods) •mRNA is NEVER polycistronic -eukaryotic RNA processing involves pre-mRNA to mature mRNA, rRNA, tRNA, micro RNA, silencing RNA we process all of them through variety of different methods and mRNA is never going to be polycistronic in eukaryotes only prokaryotes are capable of performing polycistronic mRNA transcription. •Colinearity •Coupling of transcription and translation in prokaryotes; DOES NOT occur in cells with a nucleus!! -And then also what I want you to be aware of is Collinearity of transcription is only seen in prokaryotes, it is the process of coupling transcription and translation and does not occur in eukaryotic cells because eukaryotic cells have a nucleus or a barrier between where transcription occurs where translation occurs

Main Types of RNA Polymerases

•Prokaryotes •RNA Polymerase (transcribes all types of genes) •Eukaryotes - specificity of polymerases •RNA Polymerase I •RNA Polymerase II •RNA Polymerase III -the diff types of RNA polymerases that are used in mostly eukaryotes. so in prokaryotes all transcription is done by a single RNA polymerase with those Alph,a beta, beta prime, Omega, and Sigma units. In eukaryotes genes get transcribed with different classifications of RNA polymerase so RNA polymerase 1 is going to make the large rRNA's so like on the previous slide 28S rRNA things like that. -RNA polymerase 2 is going to do mRNA. Then RNA polymerase 3 is going to do the tRNA as some of the smaller rRNA's, and then some of the micro RNA's. Don't worry about RNA polymerase four and five those are specific RNA polymerase is for plants. so you just need to know the RNA polymerase that specific prokaryotes and then 1, 2, and 3 which are specific to eukaryotes

Types of RNA Processing

•Prokaryotes and Eukaryotes •mRNA (protein-coding) •Cleavage of polycistronic sequences (PRO) -ONLY kind of processing that occurs on prokaryotes •5' Cap; polyA tail; splicing introns (EU) •rRNA •Cleavage of rRNA gene segments •tRNA •Modification of shape; excision of "excess" sequences -The types of RNA processing occur in both prokaryotes and Eukaryotes collectively. So we get minimal modification at least of mRNA or protein coding genes in prokaryotes we have cleavage of the polycistronic sequences ,this is the only kind of processing going to occur. -Eukaryotes do additional processing and they do not cleave polycistronic genes apart from each other because they don't produce polycistronic sequences. so in eukaryotes we're going to add a 5' cap you're going to add a PolyA tail and we're going to splice out introns for mRNA. -. rRNA's are both going to undergo significant amount of processing cause they're produced in kind of polycistronic units so they are produced as a series of rRNA genes that then have to get clipped part and then tRNA molecules undergo a tremendous amount of modification. Where we excise specific sequences so that the shape of the tRNA molecule becomes a mature tRNA molecule. •Small RNAs (Eukaryotes only) •miRNA •siRNA -Si and micro RNA's are the small RNA's that are going to be produced by eukaryotes only and their method of processing and necessity in the cell are going to be seen in eukaryotes

Three "parts" within a gene (DNA sequence)

•Promoter sequences (signal to start) -In each gene sequence there are three sequences in the DNA. So the first is at the beginning of the promoter sequence it is going to contain all of the signal sequences that are going to get read by the enzymes that will initiate the process of transcription. So it's called a promoter because it's going to promote transcription. •Transcribed sequence region (template) -The second part is the actual sequence that is going to be transcribed it is just the template sequence that is going to serve as the template to make the complementary base pair sequence in the RNA. So its the actual transcribed part of the sequence, the promoter does not get transcribed its just where the enzymes are going to initiate this process. •Terminator sequences (signal to end) -The 3rd part is the terminator sequence and this is just what tells the enzymes to stop transcription.

Transcription in Eukaryotes

•Protein-coding genes (mRNA) •What is needed for initiation? •Promoter -TATA Box (-25) -Up and downstream promoter elements •Control gene expression level -Enhancers -Silencers -TFIIB recognition sequence (-35) AKA CAAT box -Tx start site (+1) -Transcription factors -RNA Polymerase -initiation of transcription in eukaryotes. So essentially, we're going to be doing this in our mRNA protein coding genes, we need a bunch of sequences in the promoter and then we need a bunch of transcription factors In addition to our RNA polymerase molecule. inside the promoter we have something called the Tata box it is the same sequence as a pribnow box that we find in prokaryotes, but now the position is a little bit further upstream. -so that a box is the same sequences as the pribnow box but now it's not found at the -10 like it is a prokaryote, its found at the -25. we will also have additional upstream and downstream promoter elements that will control the level of gene expression we get from this gene, so now it's not just a question of is the transcription on or off it is also how much transcript we're getting, so are we doing a lot of transcription or are we silencing the transcription. -We have an additional sequence called the Call the TFIIB or the transcription factor two B recognition sequence this is found at the -35 usually, and you can also call it the CAAT box it is very similar to the -35 consensus sequence in prokaryotes. In addition to that we have our +1 transcription start site that's just regular in prokaryotes. We had many transcription factors in the transcription factors are specific to the gene we are actually transcribing. And we have our RNA polymerase.

Recruitment of RNA Pol II

•RNAPoly II to initiation complex •Pre-initiation complex formed! •Closed complex -So now that we have TFIID, TFIIB, TFIIA we can recruit the RNA polymerase which in eukaryotes or mRNA gene is RNA polymerase 2, to the complex. Once we do that once we have binding of all of these transcription factors and recruitment of the polymerase this is what is called the closed complex, you can also call it pre-initiation complex. Essentially what is happened is you gathered all of your enzymes and subunits to the promoter, but you have not yet unzipped the DNA. -So the reason we call it a closed complex or pre initiation complex is because the DNA molecules are still attached to each other in a double stranded fashion so we haven't made the DNA molecules single stranded yet or form that transcription bubble so we call this the close complex.

Topic Outline

•Review the central dogma of molecular biology •Overview and goal of transcription process •Transcription (tx) products •Types of genes •Gene architecture of mRNA genes (unique sequences) •Process of tx for mRNA genes -Prokaryotes (machinery and steps) -Eukaryotes (machinery and steps) •Gene architecture of rRNA and tRNA •Types of polymerases

Prokaryotes: Two Types of Termination -In prokaryotes here is two types of termination what's called row independent termination and row dependent termination.

•Rho-independent—No extra proteins required to stop transcription -Row independent termination means that the termination of transcription is pretty much spontaneous so we do not need extra proteins that will be required to stop the process of transcription. The RNA polymerase will go through the termination sequence it will hit the polyuracil sequence, and then it will just fall off the DNA. That the simplest example we have. •Rho-dependent — Uses "Rho" protein to stop RNA polymerase -Next one is a little bit more complicated it's called row dependent meaning it requires an additional protein called "row" to stop the RNA polymerase from transcribing any further. These are both in prokaryotes.

The Basics What is the product of transcription?

•Single stranded RNA molecule •Different types -mRNA -rRNA -tRNA -Small regulatory RNA (miRNA and siRNA) •Prokaryotes versus eukaryotes •Differences in processing •Occurs in different cell compartments -So the whole idea of transcription is to take a DNA sequence and to fire off a copy of RNA that is complementary to the template strand of the DNA or the exact same sequence of the coding strand of DNA. So essentially the product of transcription is going to be a single stranded RNA molecule that is put together in the exact same way that DNA would be put together. -So we're taking triphosphate nucleotides in this case they're going to be RNA nucleotides we are stringing them together making these new phosphodiester bonds, and essentially what we're getting into single stranded Poly nucleic RNA nucleic acid there several different types we will talk about mRNA, rRNA, tRNA and then two types of small regulatory RNA MI( micro RNA) SI (silencing RNA). So the similarities here are that we are going to produce one single stranded Poly nucleic acid and that Poly nucleic acid is going to be RNA so it's not DNA, DNA serves as the template but RNA is the actual product of transcription

Molecular details of the splicing of an intron

•Spliceosome cleaves 5' splice site •Bends RNA and ligates 5' nucleotide to the branch point nucleotide (5'->2' linkage!!) •Forms lariat (loop) •3' splice site is cleaved •Exons are ligated together and intron lariat is degraded -All right so this is what is happening on the molecular level, when we go to perform the splicing. so step 1 is that the spliceosome is going to recognize the 5' splice site and it is going to cleave the 5' splice site between two nucleotides, so it will cleave it between the 3' most nucleotide of the exon one and the 5' most nucleotide of the intron. Then what is going to do with that 3-5' end of the intron is a loop the molecule back around and then basically form a phosphodiester bond between the 2' OH of the 5' end phosphate and the 2' oxygen on branch point. -so essentially what is going to happen is we get this loop formation that's showing you in panel B in this top figure. the loop formation forms at what's known as the lariat or this kind of lasso formation. And then if you look at the bottom panel that like big blowup is showing you the 5'->2' linkage of this specific looping. so it's showing you that the 5' phosphate group is now joining the 2' OH oxygen that's located within the branch point adenine so it's just a phosphodiester bond, but the reason that RNA can do this is because it has an additional functional group at the 2' carbon. -. Once that happens which we form this nice little loop where we can bend the 5' end back to the bridge point, all that's left to do is chop the 3' splice site and then when we chop at 3' splice site we'd ligate the two exons together so we physically joined the 3' nucleotide from Exon 1 to the 5' nucleotide on Exon2, once we do that we get two components we get this intron that this loop or lariat shape like you can see in panel C and we get the two exons joined together. so two products of this reaction we get the lariat intron sequence and we get the two exons spliced together and then the lariat is just going to get degraded

Splicing of Introns Post-transcriptional modification Uses spliceosome

•Spliceosome is a complex of functional RNA molecules and proteins •RNAs perform the enzymatic function •Are ribozymes!!! •Bends the RNA, cuts the introns at the 5' and 3' ends AND ligates the exons together -Now we're gonna do is talk about the splicing of intron so mRNA at this point that we produced has a 5' cap it has a 3' tail and now all that's left to do in our processing is remove any of those introns sequences that exists between the exon sequences. so the machinery that we're going to use for this is called the spliceosome, it is a group of proteins that have RNA molecules kind of shoved inside the protein structure. So what the image is showing you on the right hand side as you have this like purple protein and then inside you have this secondary structures three-dimensional folded structure snRNA's. -snRNA is our functional RNA's that have enzymatic capability so in this case the ribose I'm or the spliceosome that we're going to use to get rid of the introns is not catalyzed by the protein it is catalyzed by the RNA's that are found within the protein. So these are ribozymes or enzymatic RNA's. the function of the proteins in the spliceosome is to bend the intron into this kind of loop formation such that the ribozymes or the enzymatic RNA's within the spliceosome can physically trim and cut the nucleic acid. -So what it showing you in the picture here is that we have a bunch of sub units in the spliceosome each sub unit is going to be involved in kind of bending this intron around, so that we bring the two ends of the exons together and then we cut out that interfering RNA sequence such that the two exons get ligated together. so now you can see if you compare the top panel to the bottom panel in this figure is that we don't have anymore intron between the exon sequences. So Exon one is now immediately next to Exon two and we have cut out this intron which forms a lariat. So a lariat is like a lasso right the shape of the intron that gets cut out is a lariat shaper lasso shape

Core Promoter

•TATA box functions: •Recruits the first tx factor TFIID to promoter •Initial recognition site for assembly of the tx complex •TFIID binds TATA through TBP:TATA interaction -We're going to focus on the core promoter in Eukaryotes, because it is the type of promoter that is always present for mRNA genes and then we will talk about the functions of each one of those sequences that we find within the core promoter. -So the TATA box which is found at the -25 position or 25 nucleotides extreme of the +1, is going to recruit the first transcription factor needed to assemble the RNA polymerase at the promoter, this transcription factor is called TFIID. -So TF means transcription factor, Roman numeral 2 means it's transcription factor #2, and then D is the subunit that we care about so TFIID is going to bind to the TATA box. The way that it does that is through a subunit called TBP, TBP stands for Tata binding protein. So TATA Binding Protein is found within the TFIID complex and that is how the TFIID complex binds to the TATA box.

TFIIB and TFIIA Binding

•TFIIB •Stabilizes TFIID:TATA interaction •Recruits more tx factors •Binds to -35 consensus sequence (AKA the TFIIB recognition sequence) -Once we have found TFIID to the Tata box through the TATA binding protein we're going to recruit more transcription factors. The two that I really care about are TFIIB and TFIIA these are going to stabilize the TFIID and Tata box interaction. Always you get recruitment of TFIIB and TFIIA, but sometimes you get additional proteins that are recruited this is gene specific. -The TFIIB is going to usually bind to the -35 consensus sequence but like I said before the -35 consensus sequence is not always located at the -35 so for our knowledge if you say binds to the -35 sequence that's fine that's an acceptable response if you say binds to the CAAT box or the TFIIB recognition sequence also fine, just know that it's not always at -35. That whole complex is now going to be responsible for recruiting the RNA polymerase to the promoter.

Prokaryotes

•Transcription of protein-coding genes (mRNA) •Phases •Initiation •Elongation •Termination -so in prokaryotes the process of transcribing and mRNA or protein coding gene is going to have three phases initiation, elongation, and termination. we will also see these three phases in eukaryotes although we'll have some key differences. •What is needed for initiation? •Core polymerase •Sigma factor •Promoter -Pribnow box - TATAAT - -10 position -What's needed for initiation phase of transcription for prokaryotes we're going to need what's called a core Polymerase this is going to be the enzyme complex that is used to perform transcription. we also need what's called a Sigma factor which is a type of transcription factor that is used in prokaryotes to basically complete the RNA polymerase so that can do its function. -We will need some specific DNA sequences which are found inside the architecture of the promoter element of these specific protein coding gene so a Pribnow box which is a sequence encoded TATAAT that is going to be found at the minus ten position on the coding strand of the promoter

Initiation--Eukaryotes

•Translation factors (eIFs) bind to 5' cap of mRNA •Small subunit (40s) binds to eIFs •1st tRNA binds to small subunit •Initiation complex slides down to the Kozak sequence (5'-ACCAUGG-3') •eIFs fall off, large subunit binds -The only real true difference in the process of translation between prokaryotes and eukaryotes is the process of initiation, so eukaryotes use a collection of Eukaryotic initiation factors also known as EIF's which are responsible for binding to the 5' cap and the 5' UTR and kind of coordinating the assembly of the small subunit and large subunit. so the first thing that's going to happen is we're going to bind a couple of translation factors to the 5' cap of the mRNA then the small subunit is going to be able to bind to that cap complex, and then the first tRNA carrying the methionine is going to bind to that EIF small subunit cap complex and then this full complex is going to slide down until it binds what's called the Kozak sequence. -which is basically just the sequence that located around the first AUG or the initiator codon. so in panel B of this figure what it showing is that whole entire complex including the tRNA EIF and the small subunit binding to the 5' elements lighting its way down to the Kodak sequence. once that happens the EIS fall off the large subunit binds and then we just go into the process of elongation

What types of genes exist in a genome?

•Unique sequence DNA •Gene sequences for polypeptides (i.e. mRNA genes) •Produce proteins -so here we're just going to review what types of genes exist in a genome. If you do not transcribe a region of DNA is not considered a gene, it's just considered a DNA sequence. so the two that we're going to talk about in this unit are unique sequences these are mRNA genes specifically that will go on to serve as templates to code for translated protein products. •Moderately Repetitive DNA •tRNA, rRNA, and other small RNA genes •Transposable elements (LTRs, SINEs, LINEs) -then the second group that will talk about our moderately repetitive DNA. because they do undergo at least occasional transcription GENES - DNA that is used for transcription

Termination

•What is needed for termination? •DNA signal sequences -Terminator -Complementary hairpin sequences -Poly uracil region •Additional proteins (sometimes) -So the basics for what we need in termination is we need DNA signal sequences found in the Terminator region of the actual gene architecture. So we've gone through the promoter we've gone through the sequence that actually gets transcribed, now we're moving into that third element called the promoter. -We're going to a lot of times require a complimentary hairpin sequence and then we will need a polyuracil region. In addition to that sometimes we also need other proteins to stop the process of transcription so in one of the examples will talk about the termination of transcription is pretty much spontaneous in the other two examples we're going to need some additional proteins to physically remove the RNA polymerase from the DNA molecule.

rRNA Processing- prokaryotes

•rRNA and tRNA genes are grouped in prokaryotes •One single transcript is made •Individual RNAs are methylated •Spacers are cut out and individual RNAs are released -rRNA's in prokaryotes and Eukaryotes are produced by grouping these rRNA genes together and making one transcript. so in prokaryotes we actually group rRNA's and tRNA's together and then we have to cut them apart such that they can go on to perform their function. so rRNA's and tRNA's are produced as one long transcript in prokaryotes and we have to cut them apart from one another. so one single transcript is made in the individual RNA's are going to get methylated such that the spacer regions that do not encode the functional RNA's like rRNA and tRNA are not going to get methylated. -so the methyl groups that get added to the rRNA's and tRNA's are going to serve as markers for the trimming of this transcript so as you can see in figure if it's an rRNA sequence located in this kind of dark blue color we're gonna add a bunch of methyl groups to the individual nucleotides, and then the methyl groups are going to tell the enzymes that are going to trim this molecule into its individual parts where to cut. So the 16S rRNA is the first one, tRNA is the second one, 23S RNA is the third one, 5S RNA is the last one. -So we add these little red methyl groups and what it does is it informs the enzyme where the spacers are. so the spacers are what we want to cut out and so what will do is by the time we get to the last part of this trimming, we have methylated are rRNA's the tRNA is not medullated but the spacers have all been cut out. So the spacers are cut and it releases these functional RNA's to go ahead and be released into the cell so that they can perform their function

rRNA Processing-Eukaryotes

•rRNA genes are grouped in eukaryotes (no tRNA genes) •Made as one transcript •Each RNA is methylated •Individual genes are cut out and released -eukaryotes very similar process occurs but tRNA is are not manufactured with the long rRNA transcripts. So in this case we have three rRNA's already all stuck together. We have 18S, 5.8S, and the 28S rRNA's and then we have some spacer regions between them. -so we make it as one long transcript just like we do in prokaryotes, we methylate these specific genes, and then that informs the enzymes that are doing the trimming that the regions that are not medullated or spacers and need to be cut out. So very similar process in prokaryotes as in eukaryotes