Biochem Exam 2_RNA and Protein Synthesis

5 prime capping to the mRNA

Adding a cap to the 5' end of the mRNA, very common we see a 7-methyl-guanosine as the 5 prime cap attached to the first base of the mRNA and we see a 5' to 5' triphosphate linkage between the 7-methyl-guanosine and the first base of the RNA. The first couple bases are also methylated on the 2 prime hydroxyl group.

Tetracycline

Binds the 30S ribosomal subunit Inhibits the binding of the amino-tRNA to the A site.

Streptomycin

Binds to the 30S ribosomal subunit (prokaryotes) Interferes with the initiation of protein synthesis and causes misreading of the mRNA Side effect of drug - can cause hearing loss due to selective toxicity of the auditory system cells. Used primarily for tuberculosis.

Puromyocin causes premature termination of the protein synthesis

During protein synthesis, there's several things we can do to interfer with protein synthess. One of those compounds is called puromycin which substitues for releasing factor and attaches itself to the last amino acid and causes premature truncation of the synthesizing protein. Stops protein synthesis midstream, very toxic to prokaryotes and eukarotic cells.

Shine-Dalgarno sequences precede the initiation codon

Each RNA, once it's transcribed off of the DNA, consists of (in green) AUG start codon and in addition to our start codon, we have a sequence, a consensus sequence upstream from that, that we're going to recognize, the ribosomes will attach to, then we'll find the first AUG sequence and we start creating proteins

Radio-labeled leucine added to growing immature erythrocytes. Average amount of radioactivity in hemoglobin.

Early experiments prove that protein growth is in the amino to carboxy terminus direction, amino end is the first amino acid that's put onto the growing chain.

The T-loop

Ends of telomeres are looped

Step 1 - Activation in the cytoplasm On the 3' end we add an adenine group, with an amino acid attached to it. It's also attached to a 3' hydroxyl of the tRNA

Note that the carboxyl group is attached to the 3' OH-group

The large subunit in bacteria is 50S and for eukaryotes is 60S. The small subunit is 30S and 40S respectively. There are also 21 and 33 proteins embedded in the ribosome. These are at least partly "ribosymes"

Prokaryotic composed of a 30S and a 50S, eukaryotic is same thing but slightly larger: 60S and 40S. 21 proteins found on prokaryotic and 33 proteins embedded in eukaryotic ribosome. These ribosomes do have some catalytic activity so we can call them, in part, ribosymes.

Protein Synthesis

Protein synthesis begins on free floating ribosomes in the cytoplasm. Proteins that are targeted to enter the endoplasmic reticulum will have an N-terminal sequence recognized by the SRP. Proteins that are targeted to enter the mitochondria will have an N-terminal sequence recognized by the "Tom" complex of the mitochondria. Proteins that are targeted for the nucleus will have an internal sequence call the NLS.

DNA has the ability to replicate itself, but proteins do not.

Proteins are completely dependent on being coded for by DNA.

Protein Synthesis

Proteins are synthesized starting at the N-terminal and grow towards the C-terminal

Nucleus NLS not cleaved off since proteins need to be retargeted every time the cell divides

Proteins found in nucleus also have a signal on it called an NLS (nuclear localization signal). This is not found in the leader strip but found within the protein itself. This will bind to a protein called an importin and be imported into nucleus. This can be recycled.

A common anti-biotic

Puromycin Causes premature peptide termination in bacterial cells

A 2-D view of the ribosomal RNA

Quite a bit of base pairing within the rRNA. It's still considered to be a single stranded RNA with a 3'end and a 5'end, but within that single strand there is quite a bit of intrastrand binding where you can see a lot of double stranded regions.

3. The IF2 hydrolyses its GTP and then releases from the complex along with IF1 and IF3. This then permits the 50S ribosome to assemble with the mRNA and 30S ribosome.

When this process is complete we will hydrolyze the GTP to GDP which now has cost us our 3rd high energy phosphate in this process.

mRNA isolated and hybridized to the DNA from which it was originally coded. In this hybridization there are loops (A, B, C, D, etc) that do not correspond to any of the RNA. These loops in DNA which do not hybridize to the mRNA represent DNA which is not eventually coded into the mRNA. This section of the single stranded DNA which is looping off here, represent the intron region which initially does code in the primary transcript of the mRNA, but then is spliced out before we use the mRNA for protein synthesis. Where these introns show up in figure on bottom-large % of DNA is not coding for any of the protein, it is spliced out of the RNA before we go onto protein synthesis.

mRNA hybirdized with the DNA that it came from.

Signal recognition particle targets the signal sequence into the ER

rER: in green the mRNA, in purple at position 1, a ribosome attaching to it at the first AUG sequence (reading from the 5' to the 3' direction). Once we start making a protein, it will have signal sequence at the very beginning of it. The signal sequence we recognize by a signal recognition particle (3) and it will cause the binding of this ribosome, the docking of it to the endoplasmic reticulum. That turns the endoplasmic reticulum into rough endoplasmic reticulum and then we're going to insert this protein into the lumen of the ER and this is how we create ER proteins. It's going to have that signal sequence that says this protein belongs inside the endoplasmic reticulum. If it did not have that sequence, the protein would stay out in the cytoplasm.

HIV - virus

A particularly nasty virus is the HIV virus, this map shows the gag region, the pol region, and the envelope region and also we see we have a bunch of other regions that code for additional proteins which disrupt various cellular regions within the host.

Tay Sachs

Defective hexaminidase A (lysosomal enzyme) Most common form has an insertion in exon 11 of the a-chain. Most common in Ashkenazi Jewish decent.

The tRNA

Drawing the tRNA in it's characteristic T shape, the 3' end is where the amino acid is attached to covalently and the anti codon is what's going to recognize the codon. Position 1 of the anticodon is the wobble position, this one is not as specific (where the other 2 are very specific). Also notice that the tRNA has several non-standard RNA bases in it.

Aids drugs - blocks reverse transcriptase Has 100x higher affinity for reverse transcriptase than DNA polymerase. Can be toxic to bone marrow casing anemia.

Drugs-reverse transcriptase inhibitors. Chemicals get into the RNA transcription mechanism and block the conversion to DNA. AZT is one of the most predominant drugs for the treatment of AIDs and it has an affinity for reverse transcriptase ~100 times higher than for normal DNA polymerase. Therefore it's more selective for the reverse transcriptase and has effectiveness in treating AIDs and HIV virus related illnesses.

20 different amino acids and 32 different tRNA's, yet there are 64 combinations.

In the codon table there's some redundancy, some amino acids have more than 1 codon that codes for them. There are 32 tRNA's so some of these amino acids have more than 1 tRNA coding for them.

Initiation The mRNA will attach itself to the 30s subunit in prokaryotic, 40s subunit in eukaryotic, once attached to consensus sequence, we find the first AUG and start. The IF (initiation factors)

1. Starting with the 30S subunit, initiation factors IF1 and IF3 bind. IF3 prevents the 30S from associating with the 50S The mRNA binds so that the Shine-Dalgarno sequence is just upstream from the P site and thus the start codon is at the P site

Streptomycin Causes misreading of genetic code in bacteria. Specifically, it binds to the 16S rRNA of the bacterial ribosome, interfering with the binding of formyl-methionyl-tRNA to the 30S subunit.

30S subunit consists of 16S rRNA and ribosomal proteins that bring it up to 30.

Protein Synthesis takes place on Ribosomes

70 ribosomal proteins, 20 enzymes, >12 auxiliary proteins, ~100 processing enzymes 32 tRNA's and rRNA's Ribosomes are found in cytoplasm, rER, matrix of mitochondria associated with mitochondrial membrane and chloroplasts.

Genes are have recognition sequences so the cell's machinery knows where to start reading.

And the cell has the ability to recognize whereabout these genes are found on the DNA.

Membrane bound proteins are commonly linked to farnesyl Because Ras is commonly involved in cancers, inhibitors of this linking reaction is of interest to cancer researchers.

And we can attach it to membranes, common to have membrane bound proteins, we can use a farneyl pyrophosphate as an anchor in a membrane if we want to attach a protein to a membrane.

Ubiquitin is used to turn over proteins

Another aspect of moving proteins is when we're done with a protein we need to degrade it. Proteins are going to be degraded either in a particle called a proteasome will have ubiquitin attached to them. Ubiquitin is attached only to proteins that are ready for destruction. Once ubiquitin is attached, we start digesting this protein, one amino acid at a time.

Addition of sugar groups to proteins.

Another change we can make to proteins is adding sugar groups to them. In this picture dolichol is going to be used to create a scaffolding that we create oligosaccharides on top of and move these oligosaccharides into the endoplasmic reticulum and then physically attach them to proteins to make glycoproteins.

Deamination of a C to form U, only occurs in intestine ApoB-100 is expressed in the liver. ApoB-48 uses the same gene but in the intestine the protein is truncated.

Another form of RNA processing takes place in the intestine, ApoB-100 and ApoB-48 are from the same gene, but in the intestine, after we express the RNA, we edit it to turn one of they cytosines into a uridine and therefore we turn this from a glutamine to a stop codon. Therefore we truncate a B-100 into a B-48 protein.

The Pro Opio Melano Cortin protein can be cut into numerous other functional proteins depending on which cell it is in.

Another variation, we know we can process the mRNA multiple different ways. We can cut it up and splice it diff ways. The same thing is true with different proteins. Example: Pro Opio Melano Cortin a protein, once we make it we chop it up into other possible proteins depending on which cell we're in. So we can have 1 gene coding for numerous different proteins depending on how we cut up the product.

Protein Synthesis-Step 2- Initiation (First amino acid of all proteins is either n-formal methionine for bacteria.)

Bacteria, mitochondria and chloroplasts, the first amino acid is N-formal methionine In Eukaryotes the first amino acid is methionine but from the start tRNA. There is a second tRNAmet for methonines inside the protein.

Rifampin

Bacterial RNA polymerase inhibitor Used as one of the drugs in the multidrug treatment of tuberculosis. Does not affect eukaryotic (nuclear) RNA polymerase Can also inhibit mitochondrial RNA polymerase, but a much higher dose is needed than is typically used for tuberculosis.

Diphtheria

Bacterium Corynebacterium diphtheriae Viral bacteria phage gene found in infected bacteria produce the protein toxin. The B-subunit binds to the cell membrane and facilitates the entry of the A-subunit. The A-subunit catalyzes the ADP ribosylation (from NAD) on to eukaryoic EF2. This then inhibits protein synthesis leading to cell death.

Protein Synthesis

Can account for up to 90% of ATP used by the cell. In bacteria the proteins, and machinery for proteins: ribosome and enzymes account for up about 35% of the dry weight of the cell. A typical protein of 100 amino acids takes about 5 seconds to be synthesized (quick).

Carboxylation

Can add carboxyl groups on there.

Blocks DNA transcription to RNA

Can block RNA synthesis too. Works by intercollating the GC bases and then preventing the synthesis of RNA

The 2 classes will attach itself: take an ATP at react it with an amino acid, kick of pyrophosphate and have one of the 20 amino acids attached to an AMP. Class 1 will then attach itself to the 2' OH group of the ribose sugar on the 3' end and the other one will attach itself to the 3' OH group.

Class I attaches to the 2' and Class II attaches to the 3'

Telomers are a specialized type of Reverse Transcriptase One strand has a lot of T's and G's What is interesting is the same RNA is used multiple times.

During fertilization we add telomeres to our new chromosomes using an RNA template and we build DNA from this RNA template so these telomeres are made by an enzyme called telomerase, type of naturally occurring within our own cells, of reverse transcriptase

Macrolide antibiotics

Ex. Erythromycin, clarithromycin Binds the 50S subunit Inhibits translocation

The Triplet Code t-RNA's bind to specific triplet codons.

For the 20 different, there are 32 diff tRNAs. There is some redundancy, some amino acids have more than 1 tRNA associated with them. Of these 32 different tRNAs, they have what's called an anti-codon on the adapter portion of the tRNA which recognizes the codon of the mRNA.

Rous sarcoma virus - a cancer causing virus in chickens The src gene codes for tyrosine specific kinase that affects cell division. Over expression of src will cause increased cell division.

Here is a map of a retrovirus, and we can see the gag region, the pol region, the envelope region, and now another one called the src region. This is something that codes for a tyrosine specific kinase that affects cell division and is known to cause cancer.

Thalassemias

Heterogeneous group of disorders Thalassa is Greek for sea. These disorders were first recorded in the literature around the Mediterranean Sea. These disorders are also common in Southern India and China. Alpha-thalassemias affect the alpha chain of hemoglobin and the beta-thalassemias affect the beta chain of Hemoglobin A. (Recall that HbA is a2b2) b0-thalassemia has no beta chain synthesized (homozygous). These patients would need to survive on HbA2 and HbF. b+-thalassemia has reduced beta chain synthesized. (heterozygous) One type of b-thalassemia is a nonsence mutation on codon 17 that changes a UGG to a UGA. The beta chain only has 16 AA's. Why is this?

Prokaryotic and Mitochondria systemsThe sigma subunit of RNA polymerase binds to the consensus sequence

How do we know where a gene begins? Upstream from where the first base that's coded, we have consensus sequences. In Ecoli (similar to eukaryotic) at about minus 10 base pairs (before first coded base pair we have a tataat or similar and about -35 we have another consensus sequence. And in some genes we have another one called the UP element. These consensus sequences are recognized by the sigma subunit of the RNA Polymerase and this is where the RNA polymerase binds to the gene and the sigma subunit is the one that directs which genes or family of genes is going to be recognized and then transcribed.

2. Initiation factor IF2 (which is a G-protein) that is currently bound to a GTP binds along with the start tRNAfMet

IF2 is a GTPase and it binds GTP so it's a G protein. When it goes on here, it's going to help settle in the first base which for prokaryotic will be the formal methionine. For eukaryotic it's going to be a methionine.

An RNA world. RNA bases can be made from "cyanide"???

If you take cyanide (toxic) and reflux it you can find adenosine in the pot following this chemical reaction.

Proof Reading by Aminoacyl-tRNA Synthetases Proof reading. Extremely selective, error rate quite low. Overall error rate 1/10,000.

Ile-tRNA synthetase favors Ile 200 times over valine. They differ by only a methyl group. Valine substitutes for Isoleucine only one out of every 3000 times. This is due to proof-reading and hydrolysis of the incorrect amino acid. Overall error rate is 1 out of 10,000 amino acids

Clathrin

Important protein for moving things around the cell is clathrin, the molecule that will cause vesicles to form

Genes have recognition sequences so the cell knows where to stop.

In addition to having codes as to where the gene begins, we also have codes as to where the gene terminates.

Protein Synthesis Step 1 - Activation Attachment of an amino acid to one of the 32 tRNA's

In addition to having mRNA which carries the code and the ribosome from which we do the protein synthesis, we also need 32 tRNAs that carry the 20 diff. amino acids.

Group I Intron acts as a "ribosyme to catalyze the guanosine lysis List the sequence of events for type 1 introns. Name the types of RNA that use this pathway.

In group I type neutrons, the intron itself acts as a ribosyme and catalytically cleaves itself off. It uses guanosine and it does not use ATP to drive this reaction, but it uses guanosine as a nucleophile to attack the last base of the exon and split out the first base of the intron.

In prokaryotes protein synthesis can begin even before the RNA synthesis is complete.

In prokaryotic bacteria, the mRNA even before we finish making it could already be translating proteins.

Explain the terms introns, exons, 5'-cap and poly A tail. Contrast the terms primary transcript and mature RNA

In the gene, we have stretches of DNA that don't code for proteins: Introns. These introns are transcribed into RNA when it's synthesized. Before we can make protein, we must remove introns to make mature mRNA. Heteronuclear RNA has introns that must be removed. Most of these introns act as their own enzymes and catalyze their own removal from the RNA, so the RNA removes it's own introns before it goes on to protein synthesis.

Maturation of Insulin

Insulin is a good example of what's going on in the Beta cells of the pancrease. We make our initial protein, the preproinsulin and it has a signal sequence which mmeans this will move into the endoplasmic reticulum. Once it's in there the disulfide bonds will spontaneously form and then we'll sequester them of as vesicles ready to be released when blood sugars go up. In the meantime we cut off the C-peptide making the mature insulin.

Chloramphenicol

Interferes with the tranferase activity on the 50S ribosomal subunit.

Promotes the insertion of this gene in the same gene on the other chromosome

It is thought that Type I introns might be remnants of viruses because when they're spliced out, they can be transcribed into a protein called homing endonuclease. Which is then used to take copies of this RNA, convert it into DNA and insert it into the genes of other chromosomes that wouldn't have the same intron.

alpha-Amanitin

Mushroom toxin that inhibits eukaryotic RNA polymerase. RNA polymerase II is most affected. Causes gastrointestinal disturbances, then electrolyte imbalance and fever, followed by liver and kidney disfuction. Between 40% and 90% of affected people die within a few days.

The codon is universal for almost all organisms except some variations in some mitochondria. UGA normally codes for a stop codon, but in vertebrate mitochondria, that code codes for a tryptophan instead. AUA usually isoleucine, but in vertebrates mitochondria it codes for methionine. Minor variations.

Mitochondria slightly different UGA-Trp AUA-Met CUA-Thr (Mark's)

Protein synthesis and modification takes place on and in the ER

Modifications of proteins takes place primarily in places such as Rough Endoplasmic reticulum.

(3rd type) snRNP's Small nuclear RiboNuclear Proteins Found in Eukaryotic cells and are similar to type II introns. Made from both independent RNA completed with proteins (not shown). ATP IS needed for this process

Most intron splicing takes place when the small nuclear ribonuclear proteins act as the agent or enzyme that splices out the intron. Now these are also ribosymes because they contain RNA, but they also contain protein. They do represent the vast majority of intron splicing enzymes. So we have at least a couple RNA pieces, a U1 and U2 that would be complementary to 2 regions of the RNA. The first region would be right around the section between the exon and the intron and the other one right around the adenosine will act as a nucleophile for splicing out this intron. These are very similar to Type II introns except they occur in the nuclear eukaryotic DNA, so the adenosine (bulging out) acts as the nucleophile like Type II, but we require these small nuclear ribonuclear proteins to catalyze this reaction.

I-Cell disease

Mucolipidosis II N-acetylglucosamine phosphotransferase is defective Mannose is not phosphorylated Lysosomal proteins are not recognized and are then not sent to the lysosome. Lysosomes enlarge and croud out the cytoplasm Death before the age of 8

Sickle cell anemia is a missence mutation on codon 7 (amino acid 6) that changes a GAG to GTG

Notice in the code, that we have several codons that all code for the same amino acid. Very commonly it's the 3rd letter of the codon that doesn't seem to matter. Example: CCU goes for proline, CCC, CCA, CCG all code for proline as well. CCU, CUC, CUA and CUG all code for Leucine, but what about UUA and UUG? U is not in one of the wobble positions, but we have more than 1 tRNA attached to each one of those amino acids. Therefore we can have a diff tRNA also coding for leucine. For all proteins synthesized for each of these RNAs, the first amino acid is always methionine because AUG is not only the code for methionine, it's also the start codon. There are 3 codons that represent the stop codon. When we get to the stop codon, we stop making protein and we're finished.

Some proteins have multiple subunits from multiple genes.

On the flipside, some proteins especially multiple subunit proteins can be coded for by several genes. In this example there's a 3 subunit protein and 3 different genes code for these three proteins.

Protein Synthesis Step 3- Elongation A and P sites of Ribosomes

Once first amino acid is down, we need to elongate this into the protein.

Proteins are targeted and transported to various locations in the cell. KDEL squence on a protein is found on ER proteins and receptors in the Golgi will cause these proteins to be sent back to the ER.

Once we have it made we can move these proteins inside the endoplasmic reticulum from the rough to the smooth. We can also move it onto the golgi apparatus where we can sort it. And then send it from there to other compartments such as lysosomes or we can secrete them.

Phosphorylation of Mannose targets proteins to the lysosomes

Once we put sugar groups on proteins, this could also be used as a marker for where we send them in the cell. If we have an oligosaccharide and where the terminal sugar is mannose 6 phosphate, those proteins are designated to become lysosome proteins.

Mitochondrial Cytochrome Oxidase

One odd case: There is 1 case in the mitochondrial Cytochrome oxidase where we do some editing and add 4 Us to the finished mRNA. This is going to shift the reading frame and therefore the DNA frame is not exactly a code for the protein because of this shift. Us not found in the gene.

b+-thalassemia

One type of this mutation is the AATAAA --> AACAAA This would change the AAUAAA to AACAAA -So the tail is not cut off and replaced by the poly A tail. -This decreases the expression of the beta subunit.

Methylation

Or methyl groups.

Post-translational modifications.

Phosphorylations Other chemical modifications and cross-links Di-sulfide bonds Addition of Prosthetic Groups Proteolytic cleavage. We can also put in crosslinks or prosthetic groups or we can cut up the protein.

Protein Synthesis: Step 5- Processing

Posttranslational modifications -Removal of N-terminal methionine -Removal of Signal sequences -Modification of some amino acids

Addition of phosphates - cell signaling

Process proteins we just made. We can add phosphate groups common in cell signaling

Synthesis of a tRNA

Processing of tRNAs. The tRNAs are essential for determining which specific amino acid is brought in or which codon when we do the translation of the mRNA into protein sequences. In the primary transcript, of a tRNA, we have bases at the 5' end that need to be cut off, we also have bases that need to be modified, whether it be methylation or some other modification and also removal of a section of mRNA within the tRNA almost like a intron splicing to get our final mature tRNA. In this case we see the tRNA for tyrosine.

RNA polymerase discovered in the 1960"s

RNA synthesis takes place at the DNA. The first thing we need to do is create a transcription bubble: we need to uncoil the DNA and open up the double stranded double helix to a single stranded DNA so that the RNA can be synthesized off the template strand of the DNA. We require some rewinding and unwinding both before and after the transcription bubble using the topoisomerases and gyrases. Once we have this opened up, the negative supercoiling will increase the probability of the double stranded DNA opening up and the transcription bubble is where we actually synthesize the RNA. There is no primer needed for RNA synthesis like we needed in DNA synthesis, RNA synthesis starts immediately. It will read in the 5' to 3' direction. It will be synthesized in the 5' to 3' direction, reading the template strand in the 3' to 5' direction. The coding strand is complementary, almost the same as the RNA being synthesized, but the coding strand is not actually read. The RNA-DNA hybrid is about 8 base paris long. The transcription bubble is ~17bp long. The overall footprint where the RNA polymerase binds to the DNA is ~35 bp long. The rate at which we synthesize is ~50-90 nucleotides/second.

Blocks DNA reverse transcription from RNA

Remember anti aids drug AZT and DDI are very useful in blocking DNA replication. There are drugs that can also block transcription.

Ribosomes are about 65% rRNA and 35% protein 15,000 or more ribosomes in a bacteria cell - 25% of the cell's dry weight

Ribosomes consist of a small subunit and a large subunit. We have a 30S subunit which is composed of a 16S ribosomal RNA plus proteins to make up the 30S and the 50S is made up of a 5S and a 23S ribosomal RNA plus proteins to make up a total of 50. S means sedimentation coefficient, not molecular weight, this is how dense they are when centrifuged. Larger the number, generally more dense they are.

The Second Genetic Code

Second genetic code-putting amino acid onto it's specific tRNA that it's associated. Important to have correct amino acid on correct tRNA.

Overlapping genes

Since genes can exist on both sides of the DNA double helix, we can have multiple types of RNAs and thus multiple types of proteins coding from small patches of DNA so therefore, we can have overlapping genes therefore giving different RNA sequences from this same genetic code depending on where we start and stop.

The Codon and the Anticodon

Since the tRNA binds to the mRNA in an antiparralel manner, position 1 of the codon is actually opposite position 3 of the anticodon. Therefore if position 1 of the anticodon is the wobble position, that would be position 3 of the codon that represents the wobble position. Wobble position-position where the base that's in there is not that important, the first 2 are very important in terms of binding.

Some of the modified bases found on tRNA

Some of the modified bases found on tRNA and what they look like chemically

Beta-thalassemia

Some types of the b0-thalassemia is due to an AT replacing a GT at the 5' end of the first or second intron, or at the splice junction at the 3'-end.

RNA directed RNA synthesis

Some viruses have RNA that code for proteins including a RNA depended RNA polymerase. There is no DNA intermediate. This RNA polymerase is specific for the virus's RNA and does not replicate other RNA in the cell. (There are some RNA viruses that don't even use DNA intermediates, they infect a cell with their RNA and replicate their RNA to make new virus particles).

Protein Synthesis

Step 4- Termination UAG, UAA, UGA Once synthesis of protein is finished, we come up to a stop codon, we do termination.

Lupus

Systemic lupus erythematosus is an autoimmune disease characterized by antibodies agaist chromatin, ribonucleoprotein, and cell membrane phospholipids and snRNPs snRNPs were discovered by researchers studying antibodies from Lupus patients

First experiments

Test tube experiments using synthetic poly-U produced poly-phenylalanine as a product. UUUUUUUUUUUUPhe-Phe-Phe-Phe CCCCCCCCCCCCC Pro-Pro-Pro-Pro AAAAAAAAAAAALys-Lys-Lys-Lys GGGGGGGGGGGG didn't work???

Addition of the poly A tail to the mRNAThe mRNA tail after the AAUAAA is cut off and replaced with poly A

The 2nd thing that takes place on mRNA as we process is we add a poly A tail. When the mRNA is coming off of the gene, we encounter a sequence AAUAAA. This sequence causes an enzyme complex to bind to the RNA and cleave the RNA just behind that sequence, upon cleavage of the remainder of the mRNA it is then replaced with A's so we bring in many ATPs and we put on a Poly A tail shortly after this AAUAAA sequence that has been recognized by our enzyme complex.

(4th type) A traditional enzyme certain tRNA's

The 4th type of intron removal uses an actual enzyme, an endonuclease. This is found in some tRNAs where we bring the intron out of this RNA by first cleaving it with an endonuclease. It creates a cyclic nucleotide on the 3' end of the leading exon. This I going to be cleaved further by a cyclic nucleotide phosphodiesterase, just like cAMP. On the 3' end of the latter exon, we have to prime this and energize it by first putting on a phosphate at the expense of an ATP and then adding another AMP on top of it at the expense of more high energy phosphate bonds to form an AMP at the beginning of the next exon which will then will be cleaved out when we ligate together the 2 regions of these exons.

The central dogma of molecular biology had to be rewritten with the discovery of retroviruses

The central dogma of molecular biology is that DNA can first replicate itself and that the genes on the DNA can be transcribed into RNA-transcription. Then the RNA can be translated into proteins. Reverse transcription is where RNA can be copied into DNA and this is commonly done when retroviruses infect a cell. Also RNA can be replicated into RNA, there are certain types of RNA viruses that are able to use RNA rather than DNA and then can replicate their RNA directly.

Releasing factor binds to a stop codon and terminates the growing peptide

The cost of protein synthesis 2 - ATP's (ie PPi) for the formation of the charged tRNA 1 - GTP for each tRNA placed on a codon 1 - GTP for each translocation. 122 kJ/mol for each AA. Releasing factor recognizes one of the stop codons and it will cleave the amino acid chain from the previous tRNA (instead of bringing in another amino acid).

RNA transcripts are made from genes in the DNA

The gene components of the DNA are first transcribed onto RNA

The next step is for the second AA to bind. Another G-Protein called elongation factor Tu will bind to tRNA's.

The next step is to bring in the 2nd amino acid and we have another factor called Tu which is also a G-protein and has GTP bound to it.

Synthesis of mature mRNA

The ovalbumin gene is used as an example of the RNA processing. 1st 5' capping. Followed by addition of poly A tail. Meanwhile we're also removing the introns to get the mature mRNA so the mature mRNA has it's introns removed, and a 5' cap added and a poly A tail added. The poly A tail is necessary for extending the half life of the RNA. There's RNAse enzymes in the cell which degrade RNA. Without the Poly A tail, the RNA would be degraded very quickly and we would not be able to get many proteins out of it.

RNA viruses have RNA rather than DNA If enough viruses inject enough reverse transcriptase into a cell, The RNA will be transcribed into a double stranded DNA, which can integrate into the cell's DNA

The retrovirus contains RNA genomic material rather than DNA genomic material. When it infects a cell, it injects it's RNA into the host cell and there are enzymes within the host cell that will generally destroy that RNA, however, if we have a large enough load, where enough of that RNA along with enough of the reverse transcriptase enzyme come in from the virus is able to convert that RNA into DNA, and then integrate that DNA into the host cells genome, we now can have replication of that virus.

Recall that bacterial ribosomes are free floating and eukaryotic free floating and more commonly are embedded in the RER

The ribosome consisits of the mRNA that's being transcribed and the 30S subunit which will attach itself to mRNA. Then 50S attaches itself aftwords and clamps things in place. Then tRNAs come in and read the mRNA and do the protein synthesis.

There are about 20 different enzymes that place the AA on it's tRNA. One for each AA. Some AA's have 2 tRNA's, but the same enzyme does both.

There are 2 classes of these, class I and class II. Because there are 32 tRNAs and only 20 amino acids, some of the amino acids will have more than 1 tRNA that it can be attached to.

Transposons - "jumping genes" Retrotransposons - use an RNA intermediate Defective viruses trapped in a cell? They lack the env gene.

There are 2 types of genomic material found in our genes. One is called a transposon, the other is called a retrotransposon. These are also known as jumping genes because they tend to move within our chromosomes. Further analysis finds that these things have a similarity to virus genomic material. It's thought that these things might actually have arisen from viral infections where the genes have gone bad and these are no longer able to form virus particles, but they remained inside of our genes.

Group 1 Intron Found in most cells Nuclear Mitochondrial ChloroplastrRNA, mRNA, tRNA no ATP needed

These group I intron upon cleaving the first part of the intron with a guanosine, will then catalytically direct the nucleophilic attack of the last base of the exon onto the first base of the next on, splicing out the intron very precisely.

eukaryotic

This shows the same thing happening for eukaryotic rRNA

This complex will bind to the A site along with hydrolysis of GTP and release of Tu The GDP will be displaced by a protein called Ts which will then be displaced by another GTP so the process can start over again.

This will come in, we end up hydrolyzing the GTP to GDP, but we will then place in here the 2nd amino acid.

Group II intronsmitochondria chloroplasts mRNA, fungi, algae, plants

Type II introns will also cleave themselves out autocatalytically, however they are not found in all DNA like Type I, these are found in mitochondria and chloroplasts of fungi, algea, and plants. Adenosine within the intron itself acts as the first nucleophile to splice out the first base of the intron, it then goes on to catalyze the last base of the first exon to do a nucleophilic attack on the first base of the next intron, splicing out the type II intron.

Frame Shift in Rous Sarcoma virus. Permits overlapping genes on a small piece of RNA. The gag gene is expressed more frequently than the pol gene.

Viruses generally have small genomes, but have more proteins than that small genome would normally provide for. In the viruses, they're a bit sloppy and this works to their advantage. If we read the gene directly we get the GAG protein. However if we hiccup along the reading frame and get a frame shift, we end up with another protein for the pol protein and therefore this happens about 5% of the time, spontaneously. And this is how we get more than one protein out of the same gene.

Tunicamycin(Antibiotic)blocks the first step of adding sugar groups to proteins.

We can block this process by blocking the formation of sugar groups with this antibiotic.

Eukaryotic DNA Pol I - RNA polymerase I - pre-rRNA Pol II - RNA polymerase II - mRNA - recognize the TATA box Pol III - RNA polymerase III - tRNA & 5S rRNA

We have a similar phenomenon taking place in eukaryotic DNA expression, upstream about -30 bp from beginning of first codon we have a tata box: TATAAA very common consensus sequence right upstream from the beginning of a gene, very similar between eukaryotic and prokaryotic DNA, keeping in mind that the mitochondrial DNA is very similar to prokaryotic DNA.

Introns are spans of DNA and thus RNA that does not code for the protein, so it has to be cut out.

We know that in eukaryotic DNA, we have a lot of introns coded into the RNA which needs to be spliced out. Most of these introns are self-splicing, And the mature mRNA then goes on to code for the protein

RNA synthesis take place in the nucleus Protein synthesis takes place on the ribosomes. Some ribosomes are free floating, some are attached to the endoplasmic reticulum.

When it is first synthesized at the gene, we make first heteronuclear RNA which self-splices itself to remove introns to make the mature messenger RNA. Other types of RNA are also made, the transfer RNA and the ribosomal RNA. The ribosomal RNA is used to make the ribosomes and the transfer RNA is used to translate which amino acid is going to be found for each codon on the messenger RNA. The messenger RNA will assemble with the ribosomes and the ribosome will read that messenger RNA and translate it into mature finished polypeptide.

Signal Sequence The leader sequence: 1. Starts with Met 2. is cleaved off 3. tends to be hydrophobic (yellow)

Where do we send proteins when we're done making them. Most proteins have a signal sequence. A strip at the beginning (all proteins start with methionine). But we generally cut the methionine off once it's on there. Signalling sequence can commonly be hydrophobic. Therefore it will fold up into a globular shape trying to keep water soluble amino acids on the outside and pack hydrophobic amino acids on inside.