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Molecular Biology

Robert F. Weaver

Chapter 15

RNA Processing II: Capping and Polyadenylation - all with Video Answers

Educators


Chapter Questions

04:20

Problem 1

You label a capped eukaryotic mRNA with ${ }^3 \mathrm{H}$-AdoMet and ${ }^{32} \mathrm{P}$, then digest it with base and subject the products to DEAE-cellulose chromatography. Show the elution of cap 1 with respect to oligonucleotide markers of known charge. Draw the structure of cap 1 and account for its apparent charge.

Sana Riaz
Sana Riaz
Numerade Educator
01:32

Problem 1

You are studying a virus that produces mRNAs with extraordinary caps having a net charge of -4 instead of -5 . You find these caps have the usual methylations of cap 1: the $\mathrm{m}^7 \mathrm{G}$ and the $2^{\prime}$-O-methyl on the penultimate nucleotide, but no additional methylations. Propose a hypothesis to explain the reduced negative charge and describe experiments to test your hypothesis. Describe sample positive results.

Mikayla Stephens
Mikayla Stephens
Numerade Educator
04:18

Problem 2

How do we know that the cap contains 7 -methylguanosine?

Shiksha Dutta
Shiksha Dutta
Numerade Educator

Problem 2

Design an experiment to demonstrate that CstF binds to the GU/U element of the cleavage and polyadenylation signal. How would you determine whether one or the other (GU-rich or U-rich) or both parts of this element are required for CstF binding?

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Problem 3

Outline the steps in capping.

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02:10

Problem 3

You are working in a research laboratory that studies the biochemisty of mRNA processing. You have developed an in vitro assay for both splicing and polyadenylation. You produce in vitro the following radioactive mRNA substrates (see table, next page) that either include a 5 '-cap or lack the 5 '-cap. You incubate these radioactive mRNA substrates with HeLa nuclear extract for $20 \mathrm{~min}$ at $30^{\circ} \mathrm{C}$ and electrophorese the products on a high resolution gel. You then distinguish the splicing products based on their relative sizes in the gel. You count the amount of radioactivity found in the unprocessed $m \mathrm{RNA}$ (pre$\mathrm{mRNA}$ ), the amount with intron 1 removed (splice 1 ), the amount with intron 2 removed (splice 2 ), both introns removed, and the amount of polyadenylated (poly A). You get the following results, where the number of pluses is related to the relative amount of radioactivity found in that band on the gel:
(TABLE CAN'T COPY)
Propose a hypothesis that explains all these results.

Yifan Zhou
Yifan Zhou
Numerade Educator
03:32

Problem 4

In yeast transcription complexes, the phosphorylation state of the CTD of Rpb1, as well as the spectrum of proteins associated with it, changes as transcription progresses. Currently the thought is that the shift in CTD phosphorylation from serine 5 to serine 2 may cause some RNA-processing proteins to leave the complex and possibly attract new proteins to the CTD (as depicted in Figure 15.32). Design and outline the experiments you would perform to demonstrate that the shift in CTD phosphorylation does indeed result in the release (or removal) of RNA-processing proteins as well as the addition of new RNA-processing proteins. Be sure to thoroughly explain your hypotheses to back up your experimental plans.

Rabeya Zahid
Rabeya Zahid
Numerade Educator

Problem 4

Describe and show the results of an experiment that demonstrates the effect of capping on RNA stability.

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Problem 5

Describe and give the results of an experiment that shows the synergistic effects of capping and polyadenylation on translation.

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Problem 6

Describe and give the results of an experiment that shows the effect of capping on mRNA transport into the cytoplasm.

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03:56

Problem 7

Describe and give the results of an experiment that shows the size of $\operatorname{poly}(\mathrm{A})$.

Chris Trentman
Chris Trentman
Numerade Educator
01:16

Problem 8

How do we know that poly $(A)$ is at the $3^{\prime}$-end of mRNAs?

Joanna Quigley
Joanna Quigley
Numerade Educator
02:33

Problem 9

How do we know that poly $(A)$ is added posttranscriptionally?

Shiksha Dutta
Shiksha Dutta
Numerade Educator

Problem 10

Describe and give the results of experiments that show the effects of poly $(A)$ on mRNA translatability, mRNA stability, and recruitment of mRNA into polysomes.

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01:50

Problem 11

With a simple sketch, summarize the polyadenylation process, beginning with an RNA that is being elongated past the polyadenylation site.

Rashmi Sinha
Rashmi Sinha
Numerade Educator

Problem 12

Describe and give the results of an experiment that shows that transcription does not stop at the polyadenylation site.

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Problem 13

Describe and give the results of an experiment that shows the importance of the AAUAAA polyadenylation motif. What other motif is frequently found in place of AAUAAA? Where are these motifs found with respect to the polyadenylation site?

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Problem 14

Describe and give the results of an experiment that shows the importance of the GU-rich and U-rich polyadenylation motifs. Where are these motifs with respect to the polyadenylation site?

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Problem 15

Describe and give the results of an experiment that shows the effect of the Rpb1 CTD on pre-mRNA cleavage prior to polyadenylation.

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Problem 16

Describe and give the results of an experiment that shows the importance to polyadenylation of $p o l y(A)$ polymerase and the specificity factor CPSF.

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Problem 17

Describe and give the results of an experiment that shows the effect on polyadenylation of adding $40 \mathrm{~A}$ 's to the end of a polyadenylation substrate that has an altered AAUAAA motif.

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Problem 18

Describe and give the results of an experiment that shows that CPSF binds to AAUAAA, but not AAGAAA.

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Problem 19

Describe and give the results of an experiment that shows the effects of CPSF and PAB II on polyadenylation of substrates with AAUAAA or AAGAAA motifs, with and without oligo( $A$ ) added. How do you interpret these results?

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03:15

Problem 20

Present a diagram of polyadenylation that illustrates the roles of CPSF, CStF, poly(A) polymerase (PAP), RNA polymerase II, and PAB II.

Mikayla Stephens
Mikayla Stephens
Numerade Educator
02:33

Problem 21

What part of the poly $(A)$ polymerase PAP I is required for polyadenylation activity? Cite evidence.

Shiksha Dutta
Shiksha Dutta
Numerade Educator

Problem 22

Describe and give the results of an experiment that identifies the cytoplasmic polyadenylation element (CPE) that is necessary for cytoplasmic polyadenylation.

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Problem 23

Describe and give the results of an experiment that shows that a capping enzyme binds to the RNA polymerase II CTD.

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Problem 24

Describe and give the results of a Far Western blotting experiment that shows that a component of the U1 snRNP binds to the RNA polymerase II CTD.

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Problem 25

Describe and give the results of ChIP analysis that shows: (a) that a capping enzyme associates with the RNA polymerase II CTD when it is close to the promoter but not when it is far from the promoter; and (b) that the phosphorylation state of the CTD changes as the RNA polymerase moves away from the promoter.

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Problem 26

Describe and give the results of an experiment that shows that failure of polyadenylation results in failure of proper transcription termination. Is this behavior due to failure of polyadenylation per se, or is it due to failure of cleavage of the transcript at the polyadenylation site?

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Problem 27

Describe and give the results of an experiment that indicates that transcription termination requires autocatalytic cleavage of the transcript, even as it is being elongated (cotranscriptional cleavage).

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Problem 28

Present a torpedo model for transcription termination in eukaryotes.

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