BCH5413 Exam 2

When creating a knockout animal, why do you need to engineer ES cells?

**Able to perform manipulation of the genome but the cells still grow into a whole organism. ES cells give rise to all body tissue types. iPS cells: take cells that are differentiated and cause them too undifferentiated. **ES cells = pluripotent, used to knock out genes of interest.

What are the functions of the eukaryotic core promoter.

**Attracts general transcription factors and RNAP to the transcription start site **Sets the orientation of transcription **Initiates basal level of transcription

What are the two steps to intron removal?

1. First transesterification: 2'-OH of branch site Adenine attacks the phosphodiester bond at the 5' splice site 2. Second transesterification: 3'-OH on 5' exon attacks the phosphodiester at the 3' splice site

Describe the steps necessary for transition from the PIC to the EEC.

1. RNA polymerase will begin to transcribe 2-3 nucleotide RNA transcripts2. After many cycles of the 2-3 nucleotide RNA transcripts, the B-finger of TFIIB and the switch 2 domain will stabilize a 4-5 nucleotide RNA transcript which leads to a commitment in escaping the promoter3. The RNA will grow and bumps into TFIIB trying to push it out of the transcription bubble causing stress. Stress will cause ejection of TFIIB from the polymerase/transcription bubble4. The RNA polymerase will escape from the promoter and transcription can continue downstream and enter the early elongation complex. During this stage, slippage and backtracking on DNA will happen until a longer RNA transcript is made

What is the function of the mediator?

A mediator is required for gene activation during transcription. It is a large multiprotein complex that interacts with both activators and general transcription factors including RNA polymerase. It is considered a general transcription factor because it is found at most RNA polymerase II genes. Though it is required for activation, it is not required for initiation. Ultimately, it helps provide interaction between RNA polymerase and activation proteins. It is an intermediate protein that provides a strong, stable connection for the RNA polymerase at the promoter.

What is the meaning of the term transgenic?

A transgenic organism is one whose genome has been altered by the random introduction of a foreign piece of DNA. Researchers often compare transgenic organisms to wild type or control organisms to determine the effect the transgene has. The amount of DNA inserted can vary from 1-200 base pairs

Prey Library:

All other genes (Y) in the cell that you want to investigate its interactions with Protein X. Each of the genes has its own plasmid with an activation domain connected to it.

Rho-independent termination

Also known as intrinsic termination. Needs a termination sequence, the RNA pol will transcribe this sequence and the inverted GC rich repeat can base pair with each other (3 H bonds) stable structure called hairpin structure is formed, followed by a repeat of U's. The combination of the UA plus the hairpin structure causes the RNA-DNA interactions to be weakened and then the RNA and RNA pol dissociate and transcription is terminated.

What is an enhancer? Briefly describe how a research might investigate whether a transcription element is an enhancer?

An enhancer is a tissue-specific, position- and orientation- independent DNA element that stimulates transcription. To investigate if a transcription element is an enhancer, researchers can delete areas within potential enhancer regions then radiolabel amino acids so that proteins will be labeled. When an immunoprecipitation is done and run on a gel, the area where there is a less protein is the enhancer region. This is because when the enhancer is not present (because the it was deleted), less transcription and subsequently less translation takes place and less protein is produced.

Explain 'attenuator mediated regulation' of Trp operon in 'low' levels of Trp. Be careful here. This is a complex interaction between the transcription of the leader sequence and translation of the structural proteins!

Attenuator mediated regulation: when the mRNA molecule is transcribed it can form hairpin structures. During low levels tryptophan, rna pol is going to start transcription because the repressor is not bound. It will begin to transcribe a leader sequence, a ribosome is going to bind to the leader sequence and start translation. During low levels of tryptophan it will come in contact with back to back trp codons causing translation to stall because it can't find any tryptophan AA to incorporate. It stalls on region 1, allowing for the hairpin structure to form, this does not affect RNA pol and can continue transcription. During high levels of tryptophan, the RNA polymerase is able to bind to operon and will begin transcription. Because there's a high level of tryptophan the ribosome does not stall and keeps going. Reaches region 2 and another hairpin structure forms, this hairpin structure is a terminator sequence so the RNA pol will come and terminate transcription.

Explain the phosphorylation of Pol II C- Terminal Domain (CTD) in 4 sentences. (ie. What does each phosphorylation or dephosphorylation lead to during processing?)

CTD (carboxy-terminal domain CTD of RNA polymerase II) is a target for cell modification during transcription for the purpose of encoding different signals. Serine 5 phosphorylation occurs during transcription initiation pausing. pTEb (positive transcription-elongation factor-b) then phosphorylates CTD, NELF (negative elongation factor), and DSIF (DRB sensitivity-inducing factor). Positive elongation leads to Serine 2 phosphorylation during processing.

Briefly describe how the DNA footprinting experiment works. What regions of the promoter were shown to be important for RNAP binding based upon the DNA footprint?

DNA footprinting: take DNA and run a PCR to amplify the region of the DNA of interest. Then DNA is labeled (radio label) and then either add protein of interest or have a rxn where no protein is run (important for control). Then the DNA gets cleaved/digested usually using DNase I. Then take that rxn and run it on a gel.

S1 nuclease: Explain why the lane for construct 1471 has three bands (the first at 1200bp is the full length probe)

Duplicate polyadenylation sites 240bp apart at lengths of 680 nt (to first site) and 920 nt (to second site). Construct 1471 has three bands. The band at 1200 nt is the full-length probe. The bands at lengths of 920 nt and 680 nt demonstrate that both polyadenylation signals are working.

-glucose -lactose

Even in the presence of cAMP-CAP complex, RNA synthesis is blocked when the repressor is bound to the operator. OPERON OFF

Briefly describe how fluorescence Resonance Energy Transfer (FRET) works. What type of process for promoter clearance was supported by FRET? Don't just name, describe what it means.

FRET: label on thing of interest with a donor fluorophore (green) and another things of interest with an acceptor fluorophore (red). The donor fluorophore is able to donate energy to the acceptor. If they are close to each other the donor is going to donate a lot of energy which gives fluorescence. When they are not as close, we don't get as much energy transfer, and give a decrease in fluorescence. Allows us to monitor how things are interacting with each, or how far or how close they are.

When determining if an additional copy of a gene added to a mouse genome using PCR, how would you be able to distinguish between the endogenous gene and the transgene?

For PCR, I use two primers, one inside the transgene and one outside the transgene, so I can distinguish the endogenous gene from the transgene. Since only the transgene can bind with these two primers and be amplified.

Bait plasmid

Has DNA binding domain on it. Clone in the protein of interest (X) onto the plasmid. When it is transcribed and translated, the protein of interest (X) will have the DNA binding domain on it.

What are the two components of the prokaryotic RNA polymerase holoenzyme? What is the function of each?

Holoenzyme: is the core enzyme plus accessory factor. Core enzyme: Sigma factor: important for promoter recognition. Recognizes sequences and forms protein DNA interactions in order to bring polymerase to the promoter to start transcription. Sigma70: recognizes housekeeping genes; genes that are always turned on or inducible promoter; only turned on in certain situations. Consensus sequence (perfect sequence) for sigma70: is TTGACA located in the -35 region, meaning were 35 nucleotides upstream of the transcription start site. Sigma70 also interacts with -10 region (known as pribnow box) that consensus sequence is TATAAT. The spacer in between -35 and -10 regions is also important (17 nucleotide spacer). All of this combined makes it the perfect sequence for sigma, recruiting a lot of polymerase here. Spacer is any DNA sequence of a certain length.

During PIC formation, how does TBP bin and carry out its function if an Inr and DPE are present with NO TATA box?

If TBP is associated with all of its TAFs, then TBP can make contact with the initiator element and other TAFs that are able to bind to the initiator and the downstream promoter. That is enough binding to start recruiting other elements for transcription.

Compare the E (early) spliceosome complex to the A complex.

In spliceosome assembly, the E spliceosome is the first phase of assembly. SR proteins identify and bind to exonic splice enhancers. U1 snRNP binds 5′ splice site. U2AF binds polypyrimidinic segment to identify the branch point and 3′ splice site. In the A complex, the U2 snRNP is recruited to branch point by U2AF.

In the commitment to splicing, compare the 5'SS RNA lane without and with incubation. Why are they different?

In the 5'SS RNA lane without incubation, there is a band indicating pre-mRNA, but not mRNA and in the 5'SS RNA lane with incubation, there are bands indicating both pre-mRNA and mRNA presence. These lanes are different because when the pre-mRNA is incubated with SR proteins before the addition of the competitive 5'SS RNA, the pre-mRNA gets pre-marked for splicing. The machinery has committed to splicing it into the mature mRNA form despite what happens next. Without this preincubation, the presence of the competitive 5'SS RNA inhibits the splicing reaction due to excess of splicing sites.

Briefly describe how the S1 nuclease protection assay works AND describe what it shows regarding the function of TATA box in a eukaryotic promoter.

In the S1 nuclease protection assay, hybridize the RNA that is being produced with a DNA probe labeled on 5' end. The probe needs to be hybridized close to where you think the transcription start site might be. Also, the probe need to be longer than where you think the transcription start site should be. Then, you will treat the sample with S1 nuclease. The probe will be degraded to the point where hybridization to the RNA begins. The RNA will also be degraded down to the beginning of the probe. Then, you denature the RNA, now you're only left with the probe, which is the transcription start site. You will run the probe using gel electrophoresis to determine its size. When the TATA sequence is deleted, the RNAP doesn't know where to start, so you will have multiple transcription sites.

+glucose -lactose

In the absence of lactose, the lac repressor binds the operator and transcription is blocked. OPERON OFF

Explain why the R-loop figures shown support the existence of introns.

In the experiment depicted on slide 31, the electron microscope pictures shown are images of pre-mRNA hybridization and mature mRNA hybridization. In pre-mRNA hybridization, RNA and DNA strands of the same length that are annealed to each other. Mature mRNA hybridization also demonstrates this, but with a 'loop' projecting from the middle of the RNA strand. This loop is no longer hybridized to the complementary DNA strand, which indicates that it codes for an intron sequence and it is being excised through exon splicing.

S1 nuclease: Explain why the lane for 1474 had only two bands

In this construct, the first polyadenylation site (at 680 nt) contains a deletion including the AAUAAA sequence. The other site is intact. 1474 and 1475 each only had two bands—the probe and a band the length of the non-mutated polyadenylation signal (AAUAAA). For instance, in 1474, the first signal (at 680 nt) contained a deletion of the AAUAAA signal, which is why the only band that is detected is the 920 nt band from the intact AAUAAA. This experiment demonstrates that deleting AAUAAA prevents polyadenylation just downstream of the signal. Reply

Describe the differences between repressor regulation of the Lac vs the Trp operon.

Lac operon: catabolic operon; Lactose acts as an inducer by inhibiting a repressor; the decrease of glucose and increase of lactose. The repressor protein's job is to keep the lac operon off, turning off transcription when no lactose is present. The repressor binds to the operator site and prevents the transcription of operon. When lactose is present, the repressor is no longer bound and you'll get transcription of the operon, producing the polycistronic mRNA, that encodes multiple enzymes involved in the metabolism of lactose. Lac repressor: no lactose, the lac repressor binds. Prevents polymerase form either binding to promoter or getting past the promoter clearance step. Leads to repressed transcription. Trp operon: Anabolic operon; Tryptophan acts as a corepressor. Example of attenuation, takes the fact that transcription and translation are very closely coupled together in bacteria, and it uses that to regulate the expression level of trp operon. Low tryptophan: the DNA binding domain located within the repressor is hidden when no tryptophan is present, and transcription can be carried out. High levels of transcription without an activator. High tryptophan: tryptophan itself binds to the repressor, causes conformational change in the repressor exposing the DNA binding element and binds to operator, prevents RNA pol from binding to promoter, no transcription.

When determining if an additional copy of a gene added to a mouse genome using a Southern blot, how would you be able to distinguish between the endogenous gene and the transgene?

Like PCR, I will use one probe to target the gene and another probe to target the vector in Southern Blot. When there is only one band with the size of the gene, then it is an endogenous gene. If there are two bands, one is with the size of the gene, another is the size of the vector, and it is the transgene.

What are the different ways in which nucleosome barriers to transcription can be overcome?

Nucleosomes can be modified at histone tails to loosen their association with DNA. Nucleosomes can also be remodeled by movement to another location. Lastly, nucleosomes can be partially disassembled and then reassembled after RNA pol has passed.

Describe the two mechanisms by which insulators may block the ability of enhancers to stimulate transcription of a gene.

One mechanism by which insulators may block the ability of enhancers from stimulating transcription of a gene is by having multiple insulators interact. This can occur when insulators (either on the same strand or different strands) interact and block the DNA with the enhancer from folding over to reach the promoter. Another mechanism by which insulators may block enhancers from stimulating transcription includes insulators interacting with enhancers or promoters through other proteins. This can occur if the proteins bound to the enhancer and the insulator interact with each other and therefore inhibit the transcription factor from interacting with the promoter. This may also occur if the proteins bound to the insulator and promoter interact.

In the example given, what is the importance of the CCR5 gene? Briefly describe how this technology is helpful in controlling the replication of HIV-1.

People who have a mutation of the CCR5 gene are resistant to HIV infection. The CCR5 creates a receptor on the surface of the white blood cells and that's what HIV binds too. People who have a natural mutation to this gene prevent HIV from binding, mutation does not lead to any other symptoms. The zinc finger protein is used to take an HIV infected person and cleave their DNA and allow it to heal by non-homologous end joining, making a deletion in the CCR5 gene. This makes the cells resistant to further HIV infection. Successful because HIV is an infection of the blood, therefore it is not necessary to knock out the CCR5 gene in every cell, only the cells that become infected need to be targeted. This could lead to an actual cure.

In the immunization phase, what happens to the foreign viral DNA?

Phase 1(immunization phase): how the bacterial cell makes itself immune Phase 2 (immunity phase): how it uses that to protect itself from a second infection. The foreing viral DNA is cleaved into smaller pieces called spacers. The spacers are incorporated into the CRISPR locus; part of that region of the chromosome has the spacers in it. The small pieces of viral DNA are separated from each other by black boxes called repeats.

What are the two phases of polyadenylation? Briefly describe the assay shown in lecture that supports the two phase model for polyadenylation.

Polyadenylation has 2 phases to create total 100-250 bases of poly-A tail. -First Phase(Slow adenylation , Less than 10 poly-A tail): requires ALL 4 components 1.PAP(Poly A Polymerase) 2.PAB II(Poly-A Binding Protein II) 3. Poly A signaling(AAUAAA) 4. CPSF(Cleavage and Polyadenylation Specificity Factor)) -Second Phase(Rapid adenylation, Approx after 10 poly-A tail):No need to have 1.PAP 3.Poly A signaling nor 4.CPSF. Only need PABII. Oligo(A) become primer and continue polyadenylation by PABII. Assay: 1st phase comparison is shown on the left side of gel(Red and Blue comparison). Without Poly A signaling seq(AAUAAA). There is no Poly-A tail(Blue). 2nd phase comparison. The middle segment of gel shows 2nd phase of polyadenylation. There are polyadenylation with or without poly A signaling seq. This is because the substrate is already has 40 Poly-A tail.

Explain the complementary base pairing necessary to carry out the first and second trans esterification steps in splicing.

Pre-mRNA is cleaved at the 5' end of the intron when U1 binds its complementary sequence to the 5' splice site - binds the GU. The cut end binds to the branch point downstream through pairing of guanine and adenine from the 5' end and branch point to create a loop - the lariat. The bonding of guanine and adenine is known as transesterification, where OH group on a carbon of adenine attacks the bond of guanine. During this time, U2 and U6 contribute by binding to the branch site and 3' splice site, respectively. U5 leads to the 3' end of the intron being brought into proximity, cut, and joined to the 5' end, again through transesterification. U6 binds to the lariat, U2 remains bound to the branch point, and U6 and U2 bind to each other. U5 bridges the two exons near the splice sites together. The lariat is then released with the spliced exons as well as U2, U5 and U6.

What would be the purpose of creating a reporter gene as a transgene? What types of experimental questions might you be able to ask and answer?

Reporter genes are used to investigate the expression of other genes whose expression we need to measure in a transgenic animal. Reporter genes "report" whether the gene of interest is expressed or not, or the location of gene expression. In the lecture, the example of the HKβ subunit gene is given, in which using a reporter gene, scientists want to investigate the location of expression of the HKβ subunit gene in the tissues of a transgenic mouse. LacZ gene is used as a reporter and the HKβ control region is placed in front of the LacZ gene in a plasmid vector. Now the tissues that expressed the HKβ subunit gene also expressed the reporter LacZ gene and it became easier for the scientists to locate the expression of the HKβ subunit gene because of the blue color of the tissues, expressed by the LacZ gene whose product is β-galactosidase. Other reporter genes mentioned in the textbook include the luciferase gene from firefly and the bacterial gene cat that encodes chloramphenicol acetyl transferase enzyme (CAT).

Explain "repressor mediated regulation" of the trp operon

Repressor mediated regulation combined with attenuation ensure that the operon is only turned on when trp levels are confirmed to be low because the cell doesn't want to waste energy.

Briefly describe how researchers determine that RNA Pol II has 12 subunits.

Researchers engineered one of RNA Pol II's subunits to have an epitope tag attached to it. They cloned a subunit into an expression vector next to an epitope tag so that when the subunit was expressed within the cells, all of the other subunits of RNA Pol II will form a complex with the one subunit containing the epitope tag. An antibody was then bound to the tag, the subunits were separated out and denatured by SDS. Next, the researchers used gel electrophoresis to identify the subunits, the first group of researchers identified 10 subunits. An additional group of researchers identified doublets from the gel the first group ran and identified a total of12 subunits of RNA Pol II.

Describe one line of evidence that some RNAs have the catalytic activity for splicing.

Ribozymes are RNA molecules that accelerate chemical reactions, and they're enzymes that happen to be made of RNA rather than protein. The condensation of amino acids in the peptidyl transferase center of the ribosome is catalyzed by the major RNA component of the large subunit. Similarly, the splicing of mRNA in eukaryotes is catalyzed by the U2-U6 snRNA. Thus, both the ribosome and the spliceosome are ribozymes.

Compare spliceosome splicing and group II self-splicing introns.

Spliceosome splicing is when introns are removed from the pre-mRNA by the spliceosome and the remaining exons are then spliced back together. The introns are removed by cleavage at splice sites which can be found at the 5' and 3' ends of the introns. Typically, spliceosomes remove one intron at a time. Group 2 splicing introns are self- splicing ribozymes that can splice themselves from pre- messenger RNA. The ribozymes catalyze the two transesterification reaction so that they can remove themselves. The group II introns are initiated by internal adensosine. The intron excision will occur in the absence of GTP.

Be sure to understand how alternative splicing in Drosophila can lead to sex determination

Sxl in the female completely skips the 3rd exon so this is a cassette alternative exon. Exon 3 has a stop codon in the middle. Sxl in male is non functional but in female it makes a splicing factor which enhances the splicing in the transformer as well as enhances itself. In female it is entirely excluding exon 2 which is an alternative 3' splice site. Exon 2 produces a stop codon and is not functional in males

In which organism was the CRISPR system discovered?

System of bacterial natural immunity. From studying natural immunity in bacteria.

During PIC formation, how does TBP bin and carry out its function if a TATA box is present?

TBP binds the TATA box brings the rest of TFIIB and all its associated with. If some subunits are taken away, the two TAFs can still bind to other elements next to the TATA box to help stabilize and recruit other to the promoter.

What are the two domains of TFIIB AND how to they assist in determining the position and direction of transcription?

TFIIBn and TFFBc are the two domains. TFIIBn binds to TPB and DNA while TFIIBc binds to the RNA polymerase. These two domains give TFIIB a shape and configuration that can wrap around and bind to the DNA and still reach and bind to RNA polymerase to put it into the correct position. This allows TFIIB to function as a bridge between TBP and RNA polymerase to ensure it is positioned correctly and moves in the right direction. IF TFIIB is absent, RNA polymerase could begin transcription in either direction.

What are the two functions of TFIIH?

TFIIH has kinase activity as shown by a gel mobility shift assay. Slower gel migration is observed when DABFEHPolIIA is in the presence of ATP as compared to conditions without ATP or complexes without TFIIH. The shift implies that a phosphate group is added to the complex, making it larger and therefore slower. TFIIH can also unwind DNA using helicase activity as shown by a helicase activity assay. If a short labeled strand of DNA is complexed with a longer unlabeled strand of DNA, then separation of the strands by helicase activity can be confirmed by electrophoresis. In the presence of TFIIH and ATP, a smaller fragment can be visualized denoting the labeled DNA strand separating from the larger strand. Both functions assist in transcription initiation and promoter clearance.

What is the importance of PAM sequence in CRISPR Cas9 technology?

THE PAM sequence is present in the invading viral DNA, and it is not present as part of the spacer. So the cell or the cas9 complex recognizes that the viral DNA is non-self and it will get cleaved.

What is the difference between the template strand and the coding strand of DNA? Which one has the same sequence as the transcript (with T instead of U)?

Template strand: The strand that the polymerase is going to use so it knows what nucleotides to add. Coding strand: the sequence or the code of this DNA strand will be identical to the code or the RNA. The coding strand has the thymine instead of the uracil. The coding strand and the template strand are defined for each gene, meaning that they can flip depending on which sequences tell the RNA pol which direct to go.

Briefly describe how researchers determined that there were three eukaryotic polymerases. What is the main function or each?

The 3 polymerases were first identified by Roeder and Rutter in 1969 during their experimentation with sea urchin embryos. They extracted cells and put them through a DEAE-sephadex column to separate the proteins. They then took fractions from the column and tested for incorporation of UMP. They graphed the results which showed 3 different polymerase activities. They were numbered based on when they came out of the column. They then separated the nucleus further to try to understand their roles. They isolated using gentile lysis to keep the nuclei intact and separated it further. Polymerases II and III were shown to have activity in the nucleoplasm whereas polyermase I had activity in the nucleolar fraction. Polyermase I is needed for rRNA (ribosomal RNA), polyermase II is needed for mRNA (messenger RNA) and polymerase III is needed for tRNA (translation RNA).

In addition to protecting the 5' from degradation, what are some of the 5' cap's other functions?

The 5' cap can also stimulate splicing, polyadenylation and de-capping, which is involved in the regulation of mRNA turnover. Cap binding proteins (CBPs) can also bind to the 5' cap and facilitate the transport of mRNA out of the nucleus and into the cytoplasm as well as facilitate the translation of mRNA.

What is the structure of the 5'cap?

The 5' cap contains a guanine nucleotide that is connected to the mRNA in a 5'-to-5' triphosphate linkage. Methyltransferase methylates the guanosine at the 7th position once the cap is in place, which makes it a 7-methylguanosine cap (m7G). Essentially, this 5' cap structure resembles the 3' end of RNA wherein the 5' carbon of the ribose is bonded and the 3' is left unbonded), which confers degradation resistance/resistance from 5' exonucleases.

Describe the relationship between the fact that transcription factors have domains and the development of the yeast two hybrid system.

The DNA binding domain and the activation domain in transcription factors were shown to be separate and act independently. They can be fused into two separate proteins. The Yeast Two-Hybrid System allows researchers to determine if two proteins interact with each other utilizing these two domains of transcription factors. The Yeast Two-Hybrid System requires 3 plasmids: Bait plasmid, Prey Library, and Reporter Gene Only if Protein X and Y interact with each other, will the DNA binding domain and activation domain bind together in a complex that can stabilize transcription and allow the reporter gene to be transcribed. There would be lacZ reporter gene activity indicated by blue coloration. If Protein X does not interact with Y, then the DNA binding domain binds to the UAS but there is no activation domain and therefore, no transcription of the reporter gene and no blue coloration.

During PIC formation, how does TBP bin and carry out its function if GC boxes are present without a TATA box?

The GC can bind the protein Sp1 and TFIID can bind due to the TAF #4 associating with the Sp1. This association is enough to hold the rest of TFIID in place and allows other proteins to bind and begin transcription.

Briefly describe the mechanism by with eukaryotic transcription is terminated.

The Torpedo Model demonstrates that as the mRNA is being transcribed, once the Pol II encounters the poly A site the RNA is cleaved; this leaves a mature RNA upstream that can be polyadenylated and an open end of the mRNA still attached to RNA Pol II. Axial nuclease XRN2 binds to the open end, and degrades the mRNA faster than Pol II is able to continue transcribing. XRN2 "torpedoes" RNA Pol II away from the DNA when they interact, termination transcription.

The U2snRNP is recruited to ___ by ____

The U2snRNP is recruited to branch point by U2AF.

Why is the branch point always close to the 3' splice site?

The branch point is always at a fixed close distance to the 3' splice site but the distance to the 5' splice site can vary. This is because in the first step the branch point (A) hydroxyl group at the 2' position attacks the phosphodiester bond at the 5' splice site creating a 2' to 5' connection while the 3' end is still connected to the body RNA. Then in the second step the 3' OH on the 5' splice site will attack the phosphodiester bond on the 3' splice site. You will then end up with the ligated exons and the circular intron lariat. This transesterification reaction is energetically neutral because of the configuration of the RNA, meaning how close in proximity the branch point is to the 3' splice site makes this reaction possible.

When are the capping enzymes associated with RNA polymerase?

The capping enzymes, according to the ChIP assay, are shown to be associated with RNA polymerase only at an early point of the transcription process.

What are the 2 possible repair mechanisms of a double strand break? Which one of the methods would you prefer if you wish to make a three nucleotide change in your target gene using CRISPR Cas9?

The first mechanism is to leave the double strand break alone and in some number of cells you would get non-homologous end joining. During this process you get insertions and deletions where the DNA was cleaved. The gene is likely to become nonfunctional if non homologous end joining occurs. Get a knockout of the gene of interest and allow non-homologous end joining to occur. The second mechanism is by using 2 target DNA, so 2 spacer regions that are close by to each other so that the cas9 system will bind to both regions. Allows for cleave on either side. Advantage: gives an offset double stranded break instead of a straight double stranded break, this is more efficient in homologous recombination.

In the figure of gel electrophoresis that shows the splicing reaction, why do the smaller intermediate product appear longer (higher on the gel) than the pre-mRNA?

The intermediate appears higher on the gel because it is a looped intermediate, which increases drag and moves the loop slower through the gel.

How are group I intron splicing different from both spliceosome and group II splicing?

The key difference between group I and group II introns is that in group I introns, the splicing reaction is initiated by guanosine cofactor, while in group II introns, the splicing reaction is initiated by internal adenosine.

When are the polyadenylation factors associated with RNA polymerase?

The polyadenylation factors are associated with the RNA polymerase during the entire transcription process.

How does the splicing machinery determine where to splice?

The process of pre-mRNA splicing is carried out in the cell nucleus by a large macromolecular complex called the spliceosome. Components of the spliceosome recognize special sequences at the intron ends called splice sites. The 5′ splice site (at the 5′ end of the intron) is initially bound by the U1 small nuclear RNP (snRNP), and the 3′ splice site is bound by the protein U2 auxiliary factor (U2AF). These components interact to bring the two ends of the intron together before going on to assemble the rest of the spliceosome that ultimately catalyzes the cleavage-ligation reactions.

What happens in a new infection from a previously recognized viral DNA?

The second time the bacteria encounter the same viral RNA, it will go into the cell and it will complementary base pair with the spacer because it was made from the same viral DNA. The viral DNA will then get cleaved by the CRISPRS

Reporter gene:

There is an upstream activating sequence (UAS) with a reporter gene such as LacZ. The binding of the DNA binding domain and activation domain to the UAS of this reporter gene results in gene expression. Only if Protein X and Y interact with each other, will the DNA binding domain and activation domain bind together in a complex that can stabilize transcription and allow the reporter gene to be transcribed. There would be lacZ reporter gene activity indicated by blue coloration. If Protein X does not interact with Y, then the DNA binding domain binds to the UAS but there is no activation domain and therefore, no transcription of the reporter gene and no blue coloration.

Rho-dependent termination

This is dependent on a protein called Rho. Rho has 6 identical subunits and can bind to sites within your RNA called rut sites. Once it binds, it can either thread the RNA through it or translocate up the RNA using ATP hydrolysis. After the rut site there's a termination sequence. RNA pol transcribes RNA, and eventually transcribes a rut site, which rho binds to. 60-90 nucleotides downstream of the rut site will be a termination sequence which causes the RNA pol to pause. Usually this is an inverted repeat, except there's no T rich region like in rho-independent, this causes the RNA pol to stall. Because the RNA pol stalls and rho is using ATP to thread/translocate the RNA eventually it catches up to the RNA pol and tugs on the RNA molecule. Pulling on the RNA molecule causes tension which causes RNA to dissociate.

When backtracking occurs, how is the RNA polymerase activity replaced by RNAse activity?

This occurs with the help of TFIIS. Its function is to provide Zinc ribbon that activates RNA pol II RNAse activity. The backtracked RNA is removed until the 3' end is back aligned in the active site.

Describe the method by which investigators can determine what sequences in DNA are necessary for the binding of transcription factors.

To determine where transcription factors will bind in the DNA sequence, investigators can utilize the method of DNA Footprinting. From our earlier lectures regarding DNA Footprinting, we know that protein bound DNA is generally protected from digestion from DNase (enzyme that catalyzes the cleavage of the phosphodiester bonds in DNA). As such, when DNA sequences are ran on gel electrophoresis, protein bound DNA will leave a footprint (no bands present on autography). For the experiment, investigators started with radioactively labeling DNA sequences they believed to contain transcription factor binding sites. They next incubated the DNA with protein (transcription factors) and without (control) and subjected the DNA to limited DNase I digestion to create DNA fragments. Lastly, DNA fragments are separated by size using gel electrophoresis.

Explain what part of the figure on slide 16 (separation of cleavage and polyadenylation shows that these two processes are separate steps).

To have proper polyadenylation of mRNA to create mature mRNA, it is 2 step process. Cleavage and polyadenylation(cut and polyadenylation) we need 3 components+ other small signaling factors 1. Poly(A) polymerase 2. Poly(A) signal hexamer (AAUAAA seq) 3. CPSF(Cleavage and Polyadenylation Specificity Factor) Without those 3, there is no polyadenylated mRNA and only cleaved mRNA observed( small substrate RNA). *Poly A signaling has to be very specific(AAUCAA doesn't work)

When creating knockout ES cells, why do you need to isolate cells that are sensitive to G418 AND ganciclovir?

To tell you where your transgene is being expressed! G418 neomycin analog kills cell without neomycin-resistance gene (cells with no integration) Gangcyclovir kills cells containing thymidine kinase gene (cells with non-specific recombination) Cells that grow on medium will have homologous recombination between the plasmid and WT gene

What is a transcription factory? What evidence supports the existence of transcription factories?

Transcription factories refers to the discrete sites in the nucleus of the cell, where transcription occurs. Evidence that supports transcription factories show the RNA polymerase, general transcription factors, specific transcription factors, activators and insulators are located in certain focal points inside of the nucleus. The chromosomes and the areas that need to be transcribed move into these focal points, where they gain access to the needed transcription factors- they localize to the focal points instead of transcription factors searching the DNA for the regions they need to transcribe. Genes are bale to extend out of their chromosome territories, in cis and trans to access a shared transcription factory.

Why is signal transduction an important concept in understanding when and where genes are transcribed?

Transcription factors can be modified by a series of signal transduction events caused by receptor-ligand binding on the cell membrane. In the context of transcription, when a growth factor binds to its receptor, it activates a series of signal transduction cascades that are necessary for the cell to continue its function. Enzymes that are involved in the signal transduction cascade are kinases that phosphorylate transcription factors or other compounds to get them activated. Dephosphorylation can be accomplished by phosphatase enzymes to reverse the reaction or inactivate the transcription factors as well as other compounds. It can help identify when and where a gene is transcribed by locating the position of transcription factors in a cell. The signal transduction cascade helps the transcription factors to move into the nucleus.

Describe some of the complexities of eukaryotic transcription when compared to prokaryotic transcription.

Transcription occurs in nucleus Translation occurs in cytoplasm, Transcription and translation differ in space and time. Pre-mRNA is formed and then processed to yield a mature mRNA RNA Polymerases consist of 10-17 subunits. Promoter recognition cannot be carried out by RNA polymerase alone.

Describe the steps necessary for transition from the EEC to productive elongation.

Two factors come in downstream of the TSS around +20 to +40 RNA that are associated with proximal promoter pausing. The two factors are NELF (negative elongation factor) and DSIF which are both bound to RNA polymerase II. Once the RNA transcript is long enough, NELF and DSIF help to pause RNA polymerase II in this position until it's ready to move forward. RNA capping and other signals activate phosphorylation activity by P-TEFb (positive transcription elongation factor). This has kinase activity and phosphorylates Ser 2 of CTD, NELF (which will now leave), and DSIF (which is converted from being a negative factor to being a positive factor). DSIF as a positive factor will now help recruit Elongin and ELL in order to form the fully functional, productive transcription complex. TFIIS is another factor in this time that can get RNA polymerase back on track for transcription after pausing and backtracking.

Which snRNP binds to the 5' splice site?

U1 snRNP

In the Zinc Finger approach to gene editing, what would be the equivalent of the guideRNA from CRISPR? Why? (explain the function of each)

Useful technology for gene editing, specifically for HIV infection.

Describe the evidence that suggests that life started out as an "RNA world".

We have seen exceptions to the Central Dogma and this is where this theory has been derived. The evidence shows and continues to show that RNA is capable of replication by using the catalytic functions of RNA as a ribozyme to continue make copies of itself. This occurred before present time and we continue to see evidence of this with the SARS COV-2 pandemic. (RNA virus.)

Operon

a cluster of several functionally related genes. Transcriptionally controlled by the same regulatory elements. Example: 3 genes that perform similar functions and are grouped together under the control of the same promoter, terminator, and operator sequence. The transcription machinery will transcribe all three genes into one mRNA. The mRNA is referred to as polycistronic mRNA, produced from one operon. The polycistronic mRNA contains the coding sequence for each of these genes, meaning it can produce multiple proteins

Operator

a sequence of DNA (cis-acting element) where a repressor protein binds (trans-acting element)

Where in the genome are loxP sites located in a Cre/Lox model? a. Adjacent to the Cre locus expressed in a desired cell type b. On either side of the gene that is to be knocked out c. Random sites throughout the genome

b. On either side of the gene that is to be knocked out

What is the function of TFIIB?

binds and helps to recruit RNA polymerase II to the promoter. It directs RNA polymerase II to the appropriate transcription start site.

Which of the following is the initiation event in formation of the PIC (pre initiation complex)? a. Binding of the TFII-E to PIC b. Recruitment of PolII to PIC c. Recruitment of TBP d.Helicase activity of TFII H

c. Recruitment of TBP

What is the function of cAMP in lac operon?

cAMP acts as an inducer by stimulating the activity of an activator. When there is low glucose, cAMP is going to be produced which simulates activity of activator. When there's low glucose, cAMP is produced from ATP, which can then go stimulate the activator. cAMP binds to CAP causing conformational change that allows CAP to bind to activator binding site, then recruits RNA pol, increasing the amount of RNA pol, which then increases transcription levels.

-Glucose +lactose

cAMP-CAP complex stimulates RNA pol activity and increases RNA synthesis. OPERON ON

Trans-acting factors

diffusible factors, they can function at multiple sites within a genome. Usually are DNA binding proteins such as transcription factors. Example: in diploid organisms, where we have 2 copies of each gene, if you were to mutate a transcription factor for reduced activity, it would affect transcription of everything else

Allolactose

during lactose breakdown, the lactose can be rearranged to become allolactose, which is what regulates the repressor. If there's lactose around, it will lead to production of allolactose, which can act as an inducer for transcription. It binds to sites within the lac repressor. So the repressor is no longer able to bind to the operator, so RNA pol can bind to the operator, leading to low levels of transcription. To get more transcription you need an activator protein called CAP. CAP: the accelerator pedal for transcription. Stimulates lac operon when glucose levels are low.

Cis-acting elements

elements that are closely tied to the gene, usually are DNA sequences. Example in diploid organisms: if you mutate a cis acting element, it only affects one copy of the gene, because the other copy would not be affected. Only tied to one location.

In the 3C chromatin capture experiment, if a PCR product is formed, what does that tell you about an enhancer and a promoter?

enhancers and promoters do not need to be close to each other on a DNA strand, or even on the same strand at all, to interact. When the enhancer and promoter are on separate plasmids they still bind to each other. The PCR product shows the crosslinked, or connected, sequences of the promoter and the enhancer.

Cells with thymidine kinase gene are sensitive to...

ganciclovir

+Glucose +lactose

in the presence of lactose the lac repressor is released from the operator, and transcription progresses at a slow rate. OPERON ON

Repressor

it represses or reduces transcription rates, done by clocking RNA pol from either binding to promoter or from moving past the promoter region. Repressors can respond to the environment, so they'll either bind to DNA in the presence or absence of a small molecule

Cells with the neomycin resistance gene are resistant to...

neomycin

When creating a knockout mouse, the chimeric animal is mated with one that is black and normal for the gene of interest. From this cross, three possible offspring can be produced. Explain the genetics of each of the three and what you would do next with the desired offspring.

possible progeny genomes include: A/X+ (brown, no gene of interest), A/X- (brown, gene of interest), a/X+ (black, no gene of interest) in which A/X- is the most desirable genome. I would then mate two heterozygous brown mice (X+X-) containing the gene of interest to produce a homozygous brown knockout mouse (X-X-) and screen by southern blot.

In the immunity phase, what RNAs are present in the Cas9 complex? And what is their role?

pre-CRISPR RNA-contains the spacers and repeats RNase III-cleave the RNase between the spacer and the spacer and repeat, cleaves between these spacers and repeats from the rest of the spacers and repeats tracrRNA-tracer binds to the repeat, which makes a recognition site for the RNase III

What is the function of TFIID?

starts the whole pre-initiation complex by having subunit Tata-Binding Protein (TBP) bind to the TATA box

Activators

trans-acting elements that increase transcriptional rates. Proteins that bind to cis-acting elements that are usually upstream of a promoter and they help recruit RNA pol to a promoter. Actively recruiting RNA pol to kickstart transcription. Usually binds to an activator binding site