1. RNA Polymerase

1. How long would it take for the E. coli RNA polymerase to synthesize the primary transcript for the E. coli genes encoding the enzymes for lactose metabolism, the 5,300 bp lac operon?
2. How far along the DNA would the transcription “bubble” formed by RNA polymerase move in 10 seconds?
3. Assuming that human Pol II transcribes at a similar rate, how long does it take to transcribe the 2,000,000 bp dystrophin gene?

2. Error Correction by RNA Polymerases DNA polymerases are capable of editing and error correction, whereas the capacity for error correction in RNA polymerases seems to be limited. Given that a single base error in either replication or transcription can lead to an error in protein synthesis, suggest a possible biological explanation for this difference.

3. RNA Posttranscriptional Processing Predict the likely effects of a mutation in the sequence ()AAUAAA in a eukaryotic mRNA transcript.

4. Coding versus Template Strands The RNA genome of phage Qβ is the nontemplate strand, or coding strand, and when introduced into the cell, it functions as an mRNA. Suppose the RNA replicase of phage Qβ synthesized primarily template-strand RNA and uniquely incorporated this, rather than nontemplate strands, into the viral particles. What would be the fate of the template strands when they entered a new cell? What enzyme would have to be included in the viral particles for successful invasion of a host cell?

5. Transcription The gene encoding the E. coli enzyme enolase begins with the sequence ATGTCCAAAATCGTA. What is the sequence of the RNA transcript specified by this part of the gene?

6. The Chemistry of Nucleic Acid Biosynthesis Describe three properties common to the reactions catalyzed by DNA polymerase, RNA polymerase, reverse transcriptase, and RNA replicase. How is the enzyme polynucleotide phosphorylase similar to and different from these four enzymes?

7. RNA Processing I While studying human transcription in the 1960s, James Darnell carried out an experiment that has become a classic in biochemistry, but at the time, it was incredibly perplexing. Darnell and coworkers used radioactive isotopes, such as []-labeled phosphate, to isolate and quantify RNAs from a cultured line of human cancer cells (HeLa). With this approach, they were able to identify those RNAs present in the nucleus and those present in the cytoplasm. The results were puzzling, because it was obvious that a large amount of transcription was occurring in the nucleus, but comparatively little radioactive mRNA was isolated from the cytoplasm. Moreover, the nuclear-isolated RNAs were much longer than those isolated from the cytoplasm. What can account for these observations?

8. The Transcriptome If a cell’s genome is completely known, is it possible to determine the cell’s transcriptome — the sequence of all the RNAs being produced by the cell — without additional information? Explain.

9. RNA Splicing What is the minimum number of transesterification reactions needed to splice an intron from a pre-mRNA transcript? Explain.

10. RNA Processing II Blocking the splicing of a particular pre-mRNA in a vertebrate cell also blocks a nucleotide modification reaction occurring in the rRNA. Suggest a reason for this.

11. RNA Modification I Researchers Brenda Bass and Harold Weintraub discovered double-stranded RNA-specific adenosine deaminases (ADARs) in 1987. These enzymes recognize double-stranded regions of mRNA and convert adenosine bases to inosine within these regions.

1. When ADAR encounters a double-stranded RNA substrate in water that also contains , radioactive oxygen incorporates into the RNA. Draw a reaction mechanism for ADAR that accounts for this observation.
2. After ADAR reacts with a double-stranded RNA, the RNA duplex sometimes spontaneously unwinds to form single strands. Why might this occur?
3. What is a possible consequence of adenosine to inosine conversion in the coding sequence of an mRNA?

12. Organization of RNA Processing In eukaryotes, pre-mRNA splicing by the spliceosome occurs only in the nucleus and translation of mRNAs occurs only in the cytosol. Why might the separation of these two activities into different cellular compartments be important?

13. RNA Modification II In addition to rRNAs and tRNAs, many human mRNAs also contain modified nucleotides, in particular -methyladenosine.

1. How might incorporation of -methyladenosine impact RNA processing?
2. Incorporation of -methyladenosine into the untranslated region (UTR; see Fig. 9-24) of the MAT2A transcript, which encodes a methionine adenosyltransferase, regulates the metabolism of the cofactor S-adenosylmethionine. Why would metabolism of S-adenosylmethionine be linked to -methyladenosine formation?

14. RNA Genomes RNA viruses have relatively small genomes. For example, the single-stranded RNAs of retroviruses have about 10,000 nucleotides, and the Qβ RNA is only 4,220 nucleotides long. How might the properties of reverse transcriptase and RNA replicase have contributed to the small size of these viral genomes?

15. Screening of RNAs by SELEX The practical limit for the number of different RNA sequences that can be screened in a SELEX experiment is .

1. Suppose you are working with oligonucleotides that are 36 nucleotides long. How many sequences exist in a randomized pool containing every sequence possible?
2. What percentage of these can a SELEX experiment screen?
3. Suppose you wish to select an RNA molecule that catalyzes the hydrolysis of a particular ester. From what you know about catalysis, propose a SELEX strategy that might allow you to select the appropriate catalyst.

16. Slow Death The death cap mushroom, Amanita phalloides, contains several dangerous substances, including the lethal α-amanitin. This toxin blocks RNA elongation in consumers of the mushroom by binding to eukaryotic RNA polymerase II with very high affinity; it is deadly in concentrations as low as . The initial reaction to ingestion of the mushroom is gastrointestinal distress (caused by some of the other toxins). These symptoms disappear, but about 48 hours later, the mushroom-eater dies, usually from liver dysfunction. Speculate on why it takes this long for α-amanitin to kill.

17. Detection of Rifampin-Resistant Strains of Tuberculosis Rifampin is an important antibiotic used to treat tuberculosis and other mycobacterial diseases. Some strains of Mycobacterium tuberculosis, the causative agent of tuberculosis, are resistant to rifampin. These strains become resistant through mutations that alter the rpoB gene, which encodes the β subunit of the RNA polymerase (see Fig. 26-10). Rifampin cannot bind to the mutant RNA polymerase and so is unable to block the initiation of transcription. DNA sequences from a large number of rifampin-resistant M. tuberculosis strains have been found to have mutations in a specific 69 bp region of rpoB. One well-characterized rifampin-resistant strain has a single base pair alteration in rpoB that results in a His residue being replaced by an Asp residue in the β subunit.

1. Based on your knowledge of protein chemistry, what technique could you use to detect whether a particular strain produces the rpoB mutant protein?
2. Based on your knowledge of nucleic acid chemistry, what technique could you also use to identify the mutant forms of rpoB?

19. Amputated RNAs with Prosthetic Tails The Wickens lab studies the dramatic effects of RNA processing on how a cell utilizes mRNAs, such as the c-mos (cellular mouse sarcoma) protooncogene, which controls meiosis and embryonic cell cycles in vertebrates. In frogs (Xenopus), expression of the c-mos protein is essential for maturation of oocytes after exposure to progesterone and embryo formation.

Specific RNAs can be cleaved (amputated) in Xenopus oocytes by injection of antisense DNA oligonucleotides that will hybridize to the mRNA. The formation of an RNA/DNA duplex triggers cleavage of the RNA strand by cellular RNase H. The figure below depicts the results of amputation of the c-mos mRNA after oligo injection. In this case, the oligonucleotide targeted the region of the c-mos mRNA. This region is downstream of the c-mos open reading frame (depicted by the AUG start and UAA stop codons) within the untranslated region (UTR).

After injection of a sense oligonucleotide, the oocyte maturation percentage was nearly unchanged. However, when an antisense oligonucleotide was injected, the maturation percentage was reduced by ∼60%. The c-mos mRNA was isolated from oocytes that either did or did not mature after injection of the antisense oligonucleotide. The mRNAs were analyzed by gel electrophoresis followed by northern blotting, as shown below. In a northern blot, RNAs are separated by gel electrophoresis according to their size, transferred to a membrane, and then detected by hybridization to radioactive or fluorescent complementary DNA probes.

1. What can you conclude about the importance of the c-mos mRNA poly(A) tail on oocyte maturation?
2. Why was a sense oligonucleotide used in some experiments?

   Members of the Wickens lab then decided to inject a prosthetic RNA after amputation of the c-mos mRNA. The prosthetic RNA contained the c-mos mRNA UTR as well as a region complementary to the amputated c-mos mRNA. Their observations are shown below. In these experiments, oocyte maturation was measured by the percentage of cells in which germinal vesicle breakdown (% GVBD) was observed. GVBD occurs when the oocyte nucleus (called the germinal vesicle) dissolves and the cell resumes meiosis.

   

3. How do you explain these results?

   Wickens lab scientists then tried attaching the c-mos mRNA UTR to an unrelated reporter enzyme. In this case, they used luciferase, whose enzymatic activity can easily be measured by luminescence (see Box 13-2). The total luciferase activity is also proportional to the amount of luciferase protein being produced by the cell. They tried attaching the entire c-mos mRNA UTR (mRNA1), the amputated UTR (mRNA2), or a combination of the amputated UTR with the prosthetic poly(A) tail to luciferase. High luciferase activity was observed only when either the entire c-mos mRNA UTR was used or when the prosthetic poly(A) tail was included along with the amputated UTR.

   

4. What does this experiment tell you about the likely function of the UTR and poly(A) tail for the c-mos mRNA and in oocyte maturation?
5. How might these results be useful for controlling cellular gene expression?