gen ex 3 LO review

· What is a base substitution

o A base substitution is just when you alter a single nucleotide in the DNA. Two types o Transition is when a purine is replaced with a purine (as in, A to G) or when a pyrimidine is replaced with another pyrimidine. o Transversion is when you replace a purine with a pyrimidine or vice versa. o Transition is the easier of the two. Its like becoming a man if you were born a woman versus becoming a grizzly bear if you were born a human

· Differentiate between forward mutations and reverse mutations

o A forward mutation will alter the wild-type phenotype whereas a reverse mutation will take a phenotype that is NOT the wild-type and convert it back into the wild-type which is like... pretyy wild

how is it that euk genes can be regulated via RNA processing and degradation

· Okay so as best as I can really understand from this section it's just kind of what you already knewà RNA lasts for not the longest of times so the more stable you make it the longer it'll last. How do you make it more stable? Well you give it a longer poly-A tail and blah blah blah. But if you degrade that RNA then the protein it encodes won't be produced and the information from the original gene on the DNA just won't be expressed and thus you've controlled gene expression

how many common amino acids are there

20

where are introns found

So they're super common in eukaryotes but not bacterial. We didn't THINK that prokaryotes had introns but it turns out that they are in bacteriophages, the occasional bacteria, and some archae as well as in mitochondria and chloroplasts

what is a codon

The codon is the sequence of nucleotides (mRNA) that specifies the amino acid

name the 3 steps of pre-mRNA processing

addition of 5' cap, addition of poly-A tail, and RNA splicing

· What are the general facts about DNA repair

o A lot of times they need two nucleotide strands in order to do the repair o They're super redundant à lots of systems can serve the same function which just goes to show how important it is for your DNA to be correct

· What is a regulator gene? A regulator protein? How are they related to operons?

o A regulator gene helps to control the expression of structural genes by increasing or decreasing their transcription o The regulator gene is not a part of the operon. It actually lies elsewhere and has its own promotor and everything. It produces a short strand of mRNA that produces a tiny protein called a regulator protein o The regulator protein can then bind to the portion of the operon that is called the operator and thus prevent or increase transcription

· How are riboswitches used to regulate bacterial gene expression?

o A riboswitch is essentially a regulatory sequence on the mRNA. A regulatory protein can come in and bind there and it'll alter the formation of secondary structure in the mRNA, thus affecting transcription o So if that regulatory protein is present it'll come in and bind to the riboswitch which will change the secondary structure. This change in structure will mask the ribo binding site (or just straight up terminate transcription) and thus no translation can take place o If regulator protein is absent the mRNA assumes an alternate structure that is not the one caused by the regulator protein and the ribosome binding site remains available and boom you can translate it and produce whatever protein you need

· Define suppressor mutation

o A suppressor mutation is one that is going to hide the effects of a DIFFERENT mutation + occurs at a site different from where the original mutation took place SO people that have that suppressor mutation to hide the OG mutation are DOUBLE mutants

· Talk through tRNA processing

o All tRNA molecules in both bacteria and eukaryotes will undergo some sort of processing after transcription o So basically you see modifications like splicing, trimming, and base modifications in both types of cells

· Explain the different levels of gene regulation. Which of these apply to eukaryotes and which apply to prokaryotes?

o Alteration of DNA or chromatin structuresà takes place in both. You can methylate the DNA or also pack it really tightly in the chromatin to prevent it being translated o Transcriptionà used in both. You can block or encourage transcription o mRNA processing à euk only bc bacteria really don't do any mRNA processing but we do the addition of 5' cap and poly-A tail and splice ità these affect whether or not the mRNA makes it into the cytoplasm, whether it can be translated, rate of translation, amino acid sequence, and stability of mRNA o mRNA stability, translation, and the modification of proteins after they are formed also plays a roleà probably these all occur in both

· Describe direct repair

o Altered bases are repaired directly without remvoeal of the base or the nucleotide

· Define expanding nucleotide repeat

o An expanded nucleotide repeat is basically when you have like... a LOT of a sequence that you really shouldn't have that much of. Can be a triplet sequence but doesn't have to be. o The more copies of the expanded nucleotide repeat that you have, the worse the disease or w/e those repeats cause will be § Amount of repeats also correlates with how instable the whole thing isà you're likely to develop even more repeats because the ones you already have are destabilizing your RNA THAT MUCH o Sound familiar? It's ANTICIPATION! Like when something becomes more and more severe with each generation à so when you have a couple of expanded nucleotide repeats your kid is likely to have more than you do which will exacerbate whatever the symptom of those repeats is and so on and so forth down throughout the generations

· What is a mutation

o An inherited change in genetic information (the things that inherit said change could be cells or organisms)

· what is the purpose of an insulator and how do they work?

o An insulator is a DNA sequence that blocks the effects of enhancers HOWEVER insulators are location dependent. If they're between the enhancer and the sequence that is being transcribed then it'll block the effects of the enhancer. If the insulator is nOT between the enhancer and the sequence that is being transcribed then it can have no effect on whether or not that gene does end up being transcribed

· What is the difference between intragenic and intergenic suppressor mutations

o An intragenic suppressor mutation is one that takes place within the same gene and somehow fixes the effects of the original mutation. In intergenic suppressor mutation takes place in a different gene à sometimes work by changing the way in which RNA is translated

· What is an operon? What types of cells have operons?

o An operon is a group of bacterial structural genes that are all transcribed together, as well as their promoter and additional sequences that control their transcription

· Ok so what is attenuation before we really get into all of this mess

o Attenuation is when transcription starts but is terminated before the RNA pol gets the chance to make it to the structural genes

· What are the lac operon mutations that you really should know

o B-gal mutant (lacZ-): no B-gal is produced o Permease mutants (lacY-) no permease made o Regulator gene mutant (lacI-) no regulator aka repressor produved § You can also have lacIs which is a super repressor that absolutely cannot be inactivated. It will always bind and thus will always repress o Promoter mutation (lacP-) RNA pol can't bind to the promoter o Operator mutation (lacOc): repressor can't bind to the operator

· Define and describe 3 effects that base substitutions can have on the amino acid sequence

o Base substitutions can cause missense, nonsense, or silent mutation. o A missense mutation is when the codon that is there STILL MAKES SENSE just not the kind of sense we want. So it encodes for an amino acid still, just not the right one o A nonsense mutation would change a sense codon aka one that codes for an amino acid into a nonsense codon which is one that codes for nothing and thus terminates translation o A silent mutation plays of that whole degenerate DNA thing à a silent mutation will change a codon into a different codon that luckily for you codes for the same amino acid as the original codon would've

· How is the 5' cap added and what is it's function

o Basically you're sticking an extra guanine and some methyl groups at the 5' end of the mRNA... but let's break that down just a bit o This takes place almost immediately after start of transcription and plays a pretty major role in translationà this is where the ribosome will bind when it's looking to start translation. The 5' cap also helps to increase stability and influences the removal of introns o So you've got 3 phosphate groups at the end of your strand, right? Well one of them is removed and a guanine is added with a WEIRD 5' to 5' bond. Then you add a couple of methyl groups

· What is the purpose of CRISPR RNA (cnRNA)?

o CRISPR (clustered regularly interspaces short palindromic repeats) RNA is the immune system of prokaryotic cells o They target and eliminate foreign DNA

· What is chromatin remodeling?

o Chromatin remodeling is exactly what it sounds likeà the structure of the chromatin is altered without chemically changing the structure of the histones. Basically you have a chromatin remodeling complex that comes in and binds to the DNA and moves the nucleosomes out of the way (with the help of an ATP that gets hydrolyzed) and then the transcription factors/ RNA pol can come in and bind and transcription can begin o For the chromatin remodeling complexes they can either move the nucleosome out of the way or cause conformational changes so that the DNA is more exposed

· Radiation and the types of mutation they causeo Cosmic rays, x-rays, gamma radiation

o Cosmic rays, x-rays, gamma radiation o They can alter the bases and break the phosphodiester bonds as well as causing pyrimidine dimers which then distort the shape of your DNA and block transcription which is bad of course

· What two things keep mutations low

o DNA repair and mistake detection

· What is a transposable elementà

o DNA sequences that can hop around within the genome and cause mutationsà they insert themselves into genes and disrupt that gene and can promote chromo rearrangements like deletions, duplications, and inversions o GENE MUTATIONS AND CHROMOSOME REARRANGEMENT

· 2 factors that influence mutation rates

o Differing ability to repair mutation, unequal exposure to mutagens, biological differences in spontaneous mutation

· How do intercalating agents result in frameshift mutations

o Ethidium bromide!! These basically stick themselves into the helix and distort its structure which causes all sorts of single nucleotide insertions and deletions and because they're only inserting the one nucleotide it's a little disastrous and causes some gnarly frameshifts which we LOVE

· Name some of the differences in translation initiation between bacterial and eukaryotic translation

o Eukaryotes don't have a shine Dalgarno sequenceà instead our small ribosomal subunit is going to bind to the 5' cap and the poly-A tail assists o Small ribosomal subunit is going to scan for the start codon and move down the mRNA until it finds it. After it has been found the whole process is pretty similar except that eukaryotes use way more initiation factors

· Differentiate between gene mutations and chromosome mutations

o Gene mutations are those that affect only a single gene and chromosome mutations are those that will affect either the number or the structure of the chromosomes

· What are two ways that histones can be modified? Describe the effects of these modifications and the enzymes involved

o Histone methylation: so one of the ways that you can modify your histones is by methylating them. This could either increase or decrease transcription depending on which histone/ which amino acid gets methylated. § Uses histone methyltransferases to add the methyl groups to specific histones and histone demethylase to get rid of the methyl groups o Histone acetylation: so the other way that you can modify histones are by acetylating them, so you're adding a CH3CO group to them à for the most part this is going to stimulate transcription because it destabilizes the structure of the chromatin and opens it up for transcription § Done with the help of acetyltransferase enzymes to add the acetyl groups and deacetylases to get rid of them

· How does one read the chart that specifies amino acid

o I feel like this is pretty self-explanatory as long as you know a few basic things about the chartà first thing is that they are written as they would appear in the mRNA as in the codon. So that means they're written 5' to 3'. If you're given the tRNA anticodon then you need to translate the tRNA to mRNA and potentially switch the order to 5' to 3' depending on what order they're given to you.

· What is the difference between inducible and repressible operons?

o Inducible operons are those in which transcription is normally off and you need the regulator protein to come and induce transcription. Repressible operons are those that are normally on and something must happen to repress that transcription

· What are some differences between prokaryotic and eukaryotic gene regulation

o Many bacterial and archaeal genes are organized into operons and our DNA doesn't do thatà most of our genes just have their own promoters and are transcribed separately o Chromatin structure plays a role in gene expression for eukaryotic cells because it has to be unwound before it can be transcribed o Eukaryotic cells have nuclear membranes so transcription and translation take place in two different location à basically our gene regulation is more diverse in the amount of stuff that it uses throughout the whole process

what is gene regulation

o Mechanisms and systems that control the expression of our genesà all our cells have the same information but they only use the genes that they need in the quantities that they need them, hence regulation

· Describe mismatch repair

o Mismatched bases will slightly distort DNA structure and those distorted sections need to be cut and replaced (and they are) so that everything ca work smoothly

· Differentiate between positive and negative control in operons

o Negative control is that in which the regulator protein is a repressor + prevents transcription whereas positive control is that in which the regulator protein is an activator and stimulates transcription when bound to the DNA

· Define: neutral mutation, loss-of-function mutation, gain-of-function mutation, conditional mutation, lethal mutation

o Neutral mutation: a missense mutation (remember a missense mutation is one where the codon is still a real codon it just doesn't code for the correct amino acid) that does alter the amino acid sequence but that alteration really doesn't change its function. This can happen if they new amino acid is v structurally similar to the original amino acid or if the amino acid that was switched really doesn't play an important role in the function of the protein o Loss-of-function mutations do exactly what they say they do and they cause complete or partial loss of normal protein function o Gain-of-function: these do the opposite. They cause the cell to produce a protein or other gene products whose function is NOT normally present within the cell o Conditional mutation: these are mutations that are only expressed under certain conditions (like high elevations) o Lethal mutations cause premature death. Duh.

· How would a negative inducible operon function in the presence/absence of an inducer?

o OK SO your typical regulator protein has two sites to bindà one to bind with the operator and one to bind with the inducer. Well, if the inducer isn't present then in your negative inducible operon (remember this means the reg protein is made active and thus normally represses transcription) you will see an active regulator protein chilling on the operator and preventing transcription o But introduce that inducer to the negative inducible operon and boom it binds to the open site on the reg protein, changes its shape, and thus renders it inactive. It will then fall off and transcription on that particular operon can begin

· How is genetic disease associated with defective repair pathways

o Often associated with stuff like cancer because those defects in the DNA repair mechanisms lead to an increased rate of mutation

· How do base analogs cause mispairing during replication

o Ok so a base analog is a chemical that has a really similar structure to a base so it gets incorporated bc DNA pol can't tell the difference so now it's just in your DNA but when that DNA gets used as a template strand the base analog doesn't actually code for anything and it's a little bit disastrous because it just mispairs and you get some wacky DNA being made

· How is antisense RNA used to regulate bacterial gene expression

o Ok so basically antisense RNA come in and they bind to the mRNA and thus inhibit its translationà inhibit the translation of the mRNA by blocking that ribosome binding site and boom that gene is no longer being expressed

What types of cells does pre-mRNA processing take place in and where does it occur

o Ok so in bacterial cells transcription and translation are going on at the same time so there's not a lot of time for modification, it's different in eukaryotic cells because our stuff takes place in different areas o transcription takes place in the nucleus and translation takes place in the cytoplasm

talk through translation elongation

o Ok so let's start off with the 3 available sites for translation: the A site (aminoacyl), P (peptidyl), and E (exit) sites o So when you first start off obviously you start with the fMet (because we're in bacteria) tRNA. But it's in the P site because that's just where it has to go but this is because it's the first one. Everything else is going to come into the complex at the A site o OK so elongation factor Tu (EF-Tu) goes and binds with a charged tRNA and a GTP to form a complex that will then enter the A site of the ribosome where the anticodon of the tRNA pairs with the codon of the mRNA o The GTP gets hydrolyzed and when GTP is hydrolyzed things leave so the EF-Tu and GDP leave (and are regenerated later by EF-Ts) o A peptide bond forms between the amino acids that are in the P and the A sitesà courtesy of peptidyl transferase o Ok so now the tRNA that's in the P site is going to let go of its amino acid (the peptide chain is now held to this whole structure entirely by the tRNA that's chilling in the A site) o Ribosome shifts down one codon in a process called translocation with the help of EF-G and GTP (the hydrolysis of GTP) à the tRNA are still held to the mRNA because the anticodons and codons are bound together so when the ribosome translocates the tRNA are shift so now the tRNA that was in the P site moves to the E site and immediately leaves. o A site is open so the whole process just starts all over again

· How can the lac operon be under positive control using catabolite repression

o Ok so the lac operon is negative inducible right, but it also has a form of positive control exerted on it that's called catabolite repression o So ok lets think about this huhà it's under positive control. So that means the protein that controls it is an activatorà in this case it's got a name: the catabolite activator protein o So this protein binds a little bit above the promotor of the lac operon and it reallllly helps RNA pol to bind to the promoterà without it there the RNA pol really won't bindà but in order for it to bind it has to be modified into adenosine-3',5' cyclic monophosphate aka cyclic AMP. o But in e. coli cells the concentration of cAMP is exactly inverse that of glucose (they prefer glucose, takes a lot less energy to catabolize it) but yeah when you have high levels of glucose there will be low levels of cAMP so the RNA pol can't bind to the lac operon and thus it won't be transcribed

· How is the poly-A tail added and what is its function

o Ok so this is basically the addition of 50-250 adenines after the end of transcription and it occurs in a process called polyadenylation o So you have two sequences at either end of the sequence called the polyadenylation signal --- One of these is the consensus sequence AAUAAA. This determines the point at which cleavage will take place ---Theres another sequence towards the end that is rich in uracil and guanine and determines where the cleavage of the 3' end will o cur o After the 3' end has been cleaved off adenines are added to the end to create the tail o Little side note, eukaryotic cells control how much a gene is expressed by controlling how stable it isà the stability given to a strand of mRNA depends on which protein attaches the tail and how long it is --- Also facilitates in the attachment of the ribosome to the mRNA (at 5' cap) and plays a role in RNA export out to the cytoplasm

· Differentiate between environmental induction of gene expression and tissue-specific gene expression

o Okay okay so environmental induction is basically when the environment demands that your body react in some way or another. For instance you don't have enough of thing x so you have to find a way to synthesize it o Tissue-specific gene expression is when the cells in a tissue act like they're in that tissue and produce only the stuff necessary for that function. Our neurons have all the DNA necessary for like... production of stomach acid but if they did that then that would be really freaking bad soooo they don't

· What are posttranslational modifications and why are they important

o Okay posttranslation modification does occur in both prokaryotes and eukaryotes. Sometimes they need a molecular chaperone to help them fold correctly as they are produced, other times they need to be cleaved and trimmed into something else. They can have methyl and carboxyl groups added to them. Ubiquitin ( a protein can be added). Sometimes it's chopping off the signal sequence at the amino end (the last part to be translated) that tells the cell where to go and after that you don't need it so it gets removed. Important because otherwise the proteins wouldn't work the way they're supposed to and that would be disastrous

· How is DNA methylation associated with gene regulation? What is methylated and what is the result in terms of gene regulation

o Okay so DNA methylation is of course distinct from histone methylation, yes? Yes. Okay anyways normally for DNA methylation it's when you add a methyl group to a cytosine base (usually one that is adjacent to a guanine). This massively represses transcription of that DNA. § So these areas where you see a lot of DNA methylation are called CpG islands because they contain a lot of cytosine and guanine. They're normally found near transcription start sites o CpG methylation is often associated with long term repression like what you would see with X inactivation à there's also an association between the methylation of CpG islands and the deacetylation of histones (remember that histone acetylation is going to stimulate transcription). So basically the methylation of the CpG islands represses transcription and also encourages the deacetylation of your histones which DOUBLE represses transcription

· How does strand slippage cause insertions and deletions and nucleotide repeat expansion

o Okay so basically if the strand slips just a little bit it'll mess up the transcription. So say your template strand forms a little bubble. Well it won't know it's bubbled out like that so it won't be transcribed and you'll have information missing from your new strand. And if your strand that is currently being transcribed slips then your polymerase will be tricked into thinking that it hadn't just transcribed that portion and it'll do it again thus adding extra stuffà nucleotide repeat expansion works in much the same way just with an entire codon worth of information

What do we mean when talking about the degeneracy of the genetic code?

o Okay so basically we have 4^3 different possible codons. Which means there are SIXTY FOUR codons that could code for the 20 amino acids. Three of those are stop codons but that still leaves 61 whole combos of sense codons so obviously some of the codons must code for the same amino. § We call this the degeneracy of the genetic codeà simply means that the code is redundant. You have a lot of different combos of code that could all specify the exact same amino acidà these codons are called synonymous codons because in a sense they "mean" (code for) the same thing o The other reason it's called degenerate is because there are about 30-50 types of tRNA floating around in a cell sooo some of our amino acids could be carried by more than one tRNA § Different tRNA that carry the same amino acid but have different anticodons are referred to as isoaccepting tRNA. HOWEVER (and we're going to talk about this in a second here) there are still more codons than anticodons... how to solve this issue?

· What is the relationship between how tightly DNA is packaged and whether or not transcription can occur

o Okay so basically when you've got your super tightly packed chromatin all the RNA pol/ transcription factors/ and regulator proteins can't bind so like how in the hell would they transcribe the DNA? They wouldn't. o If you want the DNA read you've gotta loosen it up a little bit

· How could an expanding nucleotide repeat be caused by strand slippage

o Okay so basically you have replication going on, right? Well say the strand that is currently being synthesized slips, forms a hairpin structure, and then manages to pair up with the template strand and transcription continues. WELL you would trick your cellular machinery into thinking that it hadn't just made those couple of triplets and boom your resultant DNA contains some additional copies of stuff

explain polyribosomes and state whether these are found in prokaryotic or eukaryotic cells

o Okay so both eukaryotic and prokaryotic cells have thisà a polyribosome is just a strand of mRNA with multiple ribosomes attached to it à each ribo attaches and moves down leaving room for another one to attach and so on

· How would a negative repressible operon function in the presence of it's product

o Okay so often times the product of the process acts as a corepressor. SO when the level of that product is really high in the cell you have enough of it that it can bind to that inactive reg protein that's floating around chilling in the cell. Once the corepressor is bound the protein becomes active and can go and repress the transcription because clearly you have enough product and the cell can kind of back off and chill

· Talk about the trp operon, how does it work, what is it an example of, what it produces?

o Okay so the trp operon is a negative repressible operonà so the negative means that the regulator protein is a repressor. Repressible means that it's normally on and the repressor is made in an inactive form. o So the trp operon makes the amino acid tryptophan, which really needs to be present in the cell. That's why its normally on and is classified as "repressible" so when tryptophan levels are normal or low the repressor is inactive and can't bind to the operator so the trp operon is transcribed and its structural genes are used to produce the amino acid tryptophan o But when tryptophan is really high tryptophan will come and bind to the inactive repressor thus making it active and then bam it can now bind to the operator and prevent transcription of the trp operon

· Differentiate between a somatic mutation and a germline mutation

o Okay so there are two big categories that you can put mutations in o The first is somatic mutation: s § omatic mutations arise in somatic tissue. If the cell survives, it'll pass its mutation on to all of its daughter cells and you'll see a population of mutant cells. So normally you see a cell mutation (aka somatic) every million cell divisions so you have a LOT of cells in you that are mutated but you normally don't see this reflected in your phenotype becuaseeeee it's just the one cell. Normally other cells will either take over for the mutated cell or the cell will just die § can lead to mosaicism o The other type is germline mutation Germline mutations are those that arise in the cells that will ultimately produce the gametes SOOOO the mutation will normally be passed down assuming viable

· Define insertions and deletions and state what type of mutation these cause

o Okay so these are exactly what they sound like they would beà an insertion is the addition of a nucleotide and deletion is the removal of one. These are actually more common than base substitution which is a little frightening given that they can lead to FRAMESHIFTS because frameshift mutations are those that alter the amino acids encoded by a sequence o HOWEVER there is such a thing as an in-frame insertion and an in-frame deletions these are indels that leave the reading frame intact either by adding or subtracting 3 (or any multiple of 3) nucleotides at a time

· Ok ok so what's the deal with wobble in the code?

o Okay so this is going to pull us back to that issue we were talking about before where there are way more codons than there are anticodons to match with them o Okay so basically what we've noticed is that a lot of the CODONS that code for the same amino acids have the same two first letters. For instance codon that is going to code for serine is going to start out with UC- and the third letter is kind of a wild card. § So what happens here is that when the first two bases of the anticodon come down they follow the Watson and crick rules and create hydrogen bonds with the base that they're supposed to pair with but the third base of that codon isn't so picky so you get some flexibility or WOBBLE in the pairing o Take away here is that the third base pairing between the codon and anticodon has some flexibility in what can pair and it's this flexibility that allows the smaller amount of anticodons to bind with the larger amount of codons provided by the mRNA

· How are TAPS, regulatory promoters, and enhancers associated with eukaryotic gene regulation. Where are these things all located with respect to the gene?

o Okok so background here: general transcription factors are a part of the basal transcription apparatus which is that complex of RNA pol and transcription factors and other proteins that carry out transcription. Basal transcription apparatus binds to the core promotor which is immediately upstream of the gene. Transcriptional regulator proteins have to come in and bind to the regulatory promotor which is upstream of the core promoter and also to enhancers which can be really far away from the gene à some of these are activators and some are repressors o TAP: transcriptional activator proteins stimulate and stabilize the basal transcription apparatus at the core promoter o oK so basically these proteins have two functions: bind at a regulatory promotor/ enhancer and interact with components of the basal transcription apparatus to influence the rate of transcription o more on this: regulatory promoters usually have several different sequences of stuff that allow different regulatory proteins to bind so you can get a cool combo of regulatory proteins o when it goes to affect the rate of transcription what the transcriptional activator does is it makes contact with the mediator which is a part of that basal transcription apparatus and destabilizes it and thus alters the rate of transcription o as for where all of this is found à the regulatory promoter is found just upstream of the core promoter which contains the TATA box, the enhancer is normally found a fair bit upstream of the actual promoters and transcription start site anddddd because the transcriptional activator protein can bind to either of these they can be found either in the regulatory promoter close to the actual sequence of DNA or they can be found upstream with the enhancer à and obviously because we're talking TAP they're going to increase the rate of transcription

· How is the trp operon controlled by attenuation

o Okok so this is like a little weird right but basically your trp operon has 4 sites.1 is complementary with 2, 2 with 3, and 3 with 4. They can pair up and create little hairpins and that change in structure is what either prevents or allows the transcription of tryptophan in the cell o When you have high levels of tryptophan 1+2 and 3+4 pair together and you end up with a hair pin (3+4) that is followed by a bunch of uracils which is an indicator of termination. So this particular structure is referred to as an attenuator because it causes attenuation aka the earlier termination of transcription o Buttt when you have low levels of tryptophan then 2+3 will bind together which DOESN'T form that hairpin/uracil structure and leave the area that contains the trp codons exposed and available for transcription. This structure is referred to as the antiterminator

· Explain how the lac operon works please and thank you

o Okokok so this is kind of a lot but basically you have permease (lacY) which helps bring the lactose into the cell and b-galactosidase (lac Z) which helps to catalyze the whole reaction. When lactose is absent, there are very few of these things made but when it's present all the production of this stuff spikes o So weird thing. Lactose isn't actually the inducer. It's allolactose. In the absence of allolactose (and lactose obv) the repressor is bound to the operator which represses transcription. o When lactose is present it'll get converted to allolactose which will bind to the repressor and make it fall off the operator. So RNA pol is no longer blocked and can come in and transcribe lacZ and lacY § But that's weird right because if you allolactose to start the production of permease and beta galactosidase then how the hell are you ever going to start transcription bc there's no permease to let the lactose into the cell and no beta gal to catalyze the reaction à production of these things never really stops even when there is a repressor bound. There's always just a little bit of stuff being made at all times

So what is the purpose of RNAi?

o RNAi is found solely in eukaryotic cells. It has two main purposes: limiting the invasion of foreign genes from viruses and such, and regulating the expression of their own genes Changes in chromo structure, translation, cell fate, and cell death

· talk through rRNA processing

o SO in bacteria: it gets methylated, cleaved, and then trimmed à produces a bunch of rRNA and some tRNA o In eukaryotes: the process is aided by snoRNA aka small nucleolar RNA which help to cleave and modify --- Much like snRNA and snRNPS that function in the splicing of pre-mRNA, snoRNA also associate with proteins to form snoRNP

· Now talk through exactly how this would work if the tryptophan level was low

o So RNA pol would begin transcribing à now you have region 1 of 5' UTR o Then a ribosome comes in and attaches to the 5' end of the mRNA that's being produced by the RNA pol. So it begins translating region 1 as RNA pol is transcribing region 2. The ribosome in this situation is going to stall at region 1 where the trp codons are because trp levels are low and you need to produce that § Meanwhile region 3 is currently being transcribed by RNA pol o Ok so region 3 is going to pair with region 2 because region 1 is busy and can't do it. Thus region 3 can't pair with region 4 and form the attenuator to stop the transcription of the trp operon so transcription continues merrily on

What is a spliceosome? Describe its makeup (what's the deal with all that RNA)

o So a spliceosome is the structure where splicing takes place. It's made up of a bunch of protein and 5 RNA molecules o Okay so the RNA. So you have these little short sequences of RNA that are called snRNA for small nuclear RNA that associate with the proteins to form snRNPs --- So what is a snRNP then? Stands for small nuclear ribonucleoprotein particle which is an absolute mouthful so you likely don't need to know all that BUT it's just that one RNA and all of its associated proteins and then five of those will make up your spliceosome --- The snRNPs are U1, U2, U4, U5, and U6 (why not U3, that's so dumb)

What is the deal with introns and exons?

o So back in the day people paired DNA with RNA and realized that DNA was longer and they were like wtfà so they realized that there were areas of DNa that code for stuff and areas that don't seem to code for anything o So the areas that code are called exons and those that don't code for anything are called introns (or intervening sequences) so all of them get turned into RNA but at some point the introns get removed and the exons are joined together

Detail how the process of RNAi works, draw the whole system and be able to tell their stories

o So basically if there is double stranded RNA in the cell it'll get chopped up by the enzyme Dicer (what a fitting name) to produce siRNA (small interfering) and miRNA (micro) o So these are a little different in that siRNA are usually exactly complementary with the target. They will pair with the mRNA and then chop it to pieces or simply inhibit translation. miRNA is not perfectly complementary and will pair and then inhibit translation § In order to do the thing where they pair, both the miRNA and siRNA will pair with a bunch of proteins in order to form a compound called RISC o After the pairing and chopping/ pairing and interfering the mRNA that doesn't belong can no longer be read and thus has been interfered with

· How does depurination cause base substitution

o So depurination is when you lose one of the purine nucleotides in your DNA strand so when replicated that apurinic strand can't provide a template for a complementary base so your transcription mechanisms really don't know what to do so they just kind of stick an adenine in there and now that's just the DNA and you have a base substitution

· Talk through what would happen in high levels of tryptophan

o So just like in situation 1 RNA pol would come and start transcribing region 1 of the DNA and making the mRNA. Then a ribosome comes in and binds to that mRNA and starts translating it as the RNA pol is down on the DNA transcribing region 2. RNA pol goes on to transcribe region 3 of the DNa and the ribosome continues on without pausing on region 1 because tryptophan levels are high so it doesn't feel the need to make that o The ribosome is now covering a portion of region 2 that it can't pair with region 3 and create that anti-terminator § So because of this region 3 is available to pair with region 4 as soon as the RNA pol is finished translating it into mRNA and once they pair they form that attenuator that will terminate transcription

describe the primary, secondary, tertiary, and quaternary

o So the primary structure of protein is simply the sequence of amino acids. o Secondary structure is the interaction of between the different amino acids the polypeptide chain will twist into the secondary structure ---- Beta pleated sheet and alpha helix are the two common ones o Those secondary structures will interact and further fold the protein into the tertiary structure o A quaternary structure can be formed through the interaction of two or more polypeptide chains

· Draw and label the general structure of an operon/ explain kind of what's up

o So the transcription of your structural genes is under the control of a single promotor which of course is upstream of said genes. RNA pol binds to the promoter and then moves down, transcribing the genes

· What are DNase I hypersensitive sites? Does transcription occur here or no?

o So these sites are found upstream of the transcription start site and get really reactive to the action of DNase 1 à basically it suggests that the chromatin in these areas adopts a more open configuration à a lot of DNase 1 hypersensitivity sites correspond to known binding sites for regulatory proteins o But yeah I don't think these areas are really transcribed

Describe how CRISPR functions

o So you have a CRISPR array that consists of a bunch of palindromic repeats that are separated by spacers (which sound really insignificant but are actually the thing you want) that are derived from the foregin DNA à cnRNA pairs up with Cas proteins to form a complex that's going to go through and destroy all that foreign shit in 3 steps o So in the first step the foreign DNA is acquired and processed. After processing that sequence is entered as a new spacer between the palindromic repeats (becomes a part of the CRISPR array). The second stage, expression, is when the CRISPR precursor RNA gets cleaved by CAS in order to form the cnRNA that contain a spacer that is homologous to the foreign DNA. That cnRNA then pairs with CAS protein to form an effector complex which can then go interfere with that DNA should it ever enter the cell again. It's basically just going to identify, bind, and then CAS cleaves that DNA so that it can't affect the cell in any way

What are the 3 consensus sequences that are required for splicing?

o So you have your 3' and 5' splice sites à these mark either end of the splice site. Changing these even a little bit would prevent splicing completely o Then you also have the branch point which is just a singular adenine nucleotide. The area around this doesn't have strong consensus so if you were to delete that adenine or mutate it to something else splicing wouldn't work o Collectively referred to as the splicing code

· Explain simultaneous transcription and translation and state what type of cell this occurs in

o Sounds vaguely terrifying and stressful but basically you have multiple ribosomes attached at the 5' end of a strand of mRNA while the 3' end is still being translated o This only happens in prokaryotes, in eukaryotic cells transcription takes place within the nucleus and translation is in cytoplasm

· What is splicing

o Splicing involves the removal of introns à takes place in the nucleus

· Difference between spontaneous and induced mutations

o Spontaneous mutations are those that occur under normal circumstances. Induced mutations are those that result from stuff like chemicals and radiation

name start and stop codons as well as what they specify

o Start codon: AUG § In prok it'll code for n-formyl methionine § In eukaryotes it'll code for methionine § This is the one that sets the reading frame because it specifies where the start is o Stop codons: UAA, UAG, UGA § There is no tRNA that is corresponds to these three codons, thus no amino acid is placed there and the sequence stopsà these are also called nonsense or termination codes

· Define structural gene, regulatory gene, and regulatory elements

o Structural gene: structural genes encode proteins that are used in metabolism or biosynthesis/ that play a structural role in the cell o Regulatory genes: the products of regulatory genes interact with other DNA sequences and affect the way that they are expressed § Little note about this: regulatory genes are often used to regulate the structural genes, but there are a few that are so necessary to general cell function that they are just expressed continually and are considered constitutive o Regulatory elements: They go out and affect the expression of the DNA sequences that they are linked to. Common in both euk and bacterial à much of the regulation of stuff within cells takes place through the action of the proteins (produced by the regulatory genes) that bind to these regulatory elements and thus control expression

· Okay so what are the 4 steps of protein synthesis (we're looking at bacterial translation) and what is tRNA charging

o The 4 steps: tRNA charging (tRNA binds to the amino acids), initiation (all the stuff needed for translation gathers together), elongation, and termination o tRNA charging is the bit where the amino acids get added to the tRNA and this is a little weird if we think back to the structure of tRNA because they all have that same end portion sticking out and that's where it binds (CCA) so like what's the deal right § specificity here comes from aminoacyl-tRNA-synthetaseà it basically recognizes the amino acid and the type of different tRNA that it can bind to o in sum: tRNA is charged (as in, has the appropriate amino acid added to it) with the assistance of aminoacyl-tRNA synthetase

what is translation and where does it occur in the cell

o Translation is basically when the mRNA is translated by the ribosome so that tRNA can come in and dump its amino acids down in the right order o So obviously this occurs outside the nucleus and within the ribosomes

· How does RNAi play a role in eukaryotic gene expression

o WELL it can come in with that RISC normally with siRNA but sometimes with miRNA and pair with an mRNA only to cleave it with Slicer o It can bind somewhere in the coding region and block translation (miRNA) o Some siRNA can come in and alter chromatin structure. They combine with proteins to form a complex called RITS which will bind to mRNA that is being transcribed and it'll attract methylation of the histone tails which causes them to bind even more tightly to the DNA o They also do Slicer independent degradation of mRNAà AU rich sequences where the miRNA can come In and bind and somehow degrade that mRNA in a way that involves Dicer and RISC

· How does unequal crossing over cause insertions and deletions

o WELL so basically when your homologous chromosomes line up they don't like up correctly so if they cross over one is going to get more information than it's going to give. The end result of that of course is that one of the sister chromatids essentially has DNA deleted and the other has a bunch of DNA inserted onto it

· What is a negative inducible operon?

o Well we know because it has the word negative in there that means its regulator protein is a repressor. Inducible means that it's normally off and you need something to induce it. Okay vaguely confusing right. So the regulator protein is a repressor but the thing is normally off. That means the protein aka the repressor is made in an active state and is normally bound to the operator which inhibits the binding of RNA pol and represses transcription.

· What is a negative repressible operon

o Well. It has negative in the name and that means that the regulatory protein that is made is going to function as a repressor of transcription. Repressible means that the operon is normally on and being transcribed. SO put those two things together and it would suggest that the regulator protein is made in its inactive form. So it's floating around but it can't stop transcription yet so transcription continues merrily along

· So knowing all of that up there, what two conditions would result in max transcription of the lac operon?

o Well. No glucose present obviously bc if there were glucose present then you wouldn't see any transcription of the lac operon whatsoever. And then you want lactose present otherwise there is only very minimal transcription of the lac operon because you don't need the products that it makes

· Explain the process of translation termination (still talking about in prokaryotes here)

o When stop codon is in the A site there's no tRNA that's going to pair up with it, instead a bunch of release factors come alongà RF1/RF2 will come into the A site depending on which stop codon is chilling in there. o RF3 comes along and joins and everything gets happily released o Just a note: remember that the termination code does not make up the 3' end of the mRNA there's that whole untranslated region chilling at the end that helps with stability and stuff like that

Talk through the process of splicing. Occurs where?

o Whole process occurs within the splice site. So you've got your intron. The pre-mRNA gets cut at the 5' splice site first and then that 5' end of the intron is going to fold back and attach to the adenine at the branch point in order to form a lariat. o Step 2: the 3' splice site is cut and the 5' of exon 2 and 3' of exon 1 will come together so that you've got a connected strand of stuff that you want expressed. Now your little intron lariat is just floating around and the exon gets exported to the cytoplasm where it gets translated o In more detail in terms of what happens at the very beginning --- snRNP U1 attaches to the 5' splice site and then U2 attaches to the branch point. A complex of U4/5/6 comes together and joins and then step 1 begins with the 5' end being spliced

· explain the process of translation initiation in prokaryotes

o okay so IF3 is going to bind to the small subunit of the ribosomeà this is going to prevent the large subunit from binding to it and also allow the small subunit to bind to the mRNA. § So basically what's happening with the mRNA binding to the complex of the small ribosomal subunit/ IF3à the rRNA of the small subunit is complementary to the Shine-Dalgarno sequence that can be found on that bacterial mRNA and that's how it attaches o A charged tRNA (as in, one that already has its amino acid) forms a complex with IF2 and GTP o That complex attaches to the initiation/start codon o IF1 will bind to the small subunit of the ribo/ IF3 complex o GTP is hydrolyzed to GDP and when GTP is hydrolyzed shit leaves so we see all 3 initiation factors leave o The large subunit comes down like the top of a rice cooker and boom initiation completeà ribo is assembled on the mRNA and the first tRNA has been attached to the start codon

talk through the basic structure of tRNA

o okay so in terms of primary structure they of course have the four regular bases but they also have a couple of modified bases (by tRNA modifying enzymes) o their secondary structure however is the same. They all have complementary sequences that result in the formation of a cloverleaf structureà there are four arms. --- Three of those arms have loops at the end where no complementary sequences lie, but the one arm at the top (acceptor arm) doesn't. acceptor arm instead has a stem, the longer arm ALWAYS ends in CCA (so obv this doesn't play any sort of role in deciding which amino acid the tRNA will pair with) --- The bottom arm is referred to as the anticodon arm and the nucleotides here pair up with the corresponding codon on the mRNA so that the amino acids end up in the correct order o Cloverleaf normally folds on itself to form L shaped structure

· how are silencers, regulatory promoters, and repressors associated with eukaryotic gene regulation. Where are these things located in relation to the gene

o okay so you can have regulator proteins that function as repressors. This we know. So the repressors can either bind to the regulatory promoter or they can bind to silencers which are a lot like enhancers in that they are not located near the actual gene o unlike bacterial repressors eukaryotic repressors really don't directly block RNA pol but they might get in the way of transcriptional activators, or bind near the binding site and prevent activators from interacting with the basal transcription apparatus, or they might directly interfere with assembly of basal transcription apparatus

define rRNA

o rRNA is the type of RNA that makes up the ribosomes à ribosomal RNA

basic structure of a ribosome

o ribosomes consist of two subunitsà a large and a small subunit. These subunits are made up of rRNA and a bunch of proteins

function of tRNA

o tRNA or transfer RNA is the link between mRNA and protein. Every tRNA attaches itself to a specific type of amino acid (they can all only attach themselves to one specific type) and then adds them together with the help of the ribosome as per the instructions given in the mRNA

talk through alternative splicing

o this is one of the alternative processing pathways for pre-mRNA. Basically the same strand of pre-mRNA can be spliced in different ways that would yield different amino acid sequences and thus different proteins o so basically you could splice out some exons or leave them in and that would change the final products

talk through multiple 3' cleavage sites

o this is the other type of alternative processing for pre-mRNA o okok so think back to the whole poly-A tail thing. Basically pre-mRNA can contain multiple 3' cleavage sites so you can chop it one way or another and add the poly-A tail at different locations and thus alter the m-RNA that is produced. This may or may not produce a different protein but the difference in length definitely affects the stability/ longevity of the mRNA protein