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Molecular Cell Biology Lodish 6th Edition Test Bank

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Post-transcriptional Gene Control

PART A: Linking Concepts and Facts

8.1 Processing of Eukaryotic Pre-mRNA

1. The consensus sequence for poly(A) addition is

a. the site of poly(A) tail addition.

b. AAUAAA

c. downstream of the cleavage site.

d. none of the above

Ans: b

2. Histone mRNAs lack

a. poly(A) tails

b. introns

c. a 3´UTR

d. all of the above

Ans: d

3. Which of the following cis and trans elements function together in a biological process?

a. Nut and N

b. AAUAAA and CPSF

c. TAR and TAT

d. all of the above

Ans: d

4. Which process involves two transesterification reactions?

a. splicing

b. RNA editing

c. capping

d. nuclear transport

Ans: a

5. Splice sites in pre-mRNA are marked by two universally conserved sequences contained

a. in the middle of the intron

b. at the ends of the exons

c. at the ends of the introns.

d. none of the above

Ans: c

6. Splicing joins

a. two intron sequences

b. two polypeptides

c. two DNA molecules

d. two exon sequences

Ans: d

7. The branch point A residue involved in lariat formation is part of the

a. intron

b. exon.

c. 5´'UTR

d. 3´UTR.

Ans: a

8. Indicate the order in which the following steps occur in the production of a mature mRNA.

a. initiation of transcription, splicing, addition of 5´' cap, addition of poly(A) tail, transport to cytoplasm

b. initiation of transcription, addition of 5´ cap, splicing, addition of poly(A) tail, transport to cytoplasm

c. initiation of transcription, addition of poly(A) tail, addition of 5´ cap, splicing, transport to cytoplasm

d. initiation of transcription, addition of 5´ cap, addition of poly(A) tail, splicing, transport to cytoplasm

Ans: d

9. Components of the spliceosome include

a. a single snRNP containing several different snRNAs

b. proteins that react immunologically with the sera of patients with systemic lupus erythematosus

c. U5 snRNA, which interacts with the 5´ splice site in pre-mRNA

d. all of the above

Ans: b

8.2 Regulation of Pre-mRNA Processing

10. Sex lethal protein in Drosophila can best be described as a(n)

a. splicing regulatory factor.

b. RNA editing factor

c. transcription factor

d. all of the above

Ans: a

11. Differential splicing of Drosophila slo RNA produces

a. various Slo proteins with different biological functions

b. various Slo proteins with identical biological functions

c. various slo mRNAs with differential stability.

d. various slo mRNAs with new nonsense codons

Ans: a

12. Which of the following does not require protein enzymes?

a. RNA editing

b. excision of group II introns

c. transsplicing

d. excision of group III introns

Ans: b

13. RNA editing is

a. post-transcriptional alteration of sequences in mRNAs

b. pretranscriptional alteration of sequences in RNAs.

c. post-transcriptional joining of two RNA molecules

d. none of the above

Ans: a

8.3 Transport of mRNA Across the Nuclear Envelope

14. Which of these events does not occur within the nucleus?

a. RNA editing in mammals

b. RNA capping

c. polyadenylation

d. RNA editing in protozoans

Ans: d

15. Which type of RNA participates in nuclear export of mRNA?

a. snRNA

b. hnRNA

c. tRNA

d. rRNA

Ans: b

16. Transport of unspliced HIV mRNA from the nucleus to the cytoplasm of host cells is promoted by a virusencoded protein named

a. Tat.

b. Rev

c. nucleoplasmin.

d. Ran

Ans: b

8.4 Cytoplasmic Mechanisms of Post-transcriptional Control

17. microRNAs play a key role in which of the following?

a. translational repression

b. viral RNA degradation

c. RNA interference

d. all of the above

Ans. a

18. Which of the following can affect the cytoplasmic location of mRNAs?

a. the cytoskeleton

b. cytochalain B

c. polyribosomes

d. all of the above

Ans: d

19. Which of the following does not part in the degradation process of eukaryotic mRNAs?

a. capping

b. endonucleolytic cleavage

c. exonucleolytic decay

d. poly(A) shortening

Ans: a

8.5 Processing of rRNA and tRNA

20. Synthesis of pre-rRNA occurs in the

a. nucleolus

b. endoplasmic reticulum

c. extranucleolar area of the nucleus

d. cytosol

Ans: a

21. The 45S pre-rRNA molecule

a. can organize a nucleolus when present in a single copy

b. is encoded by genes that are tandemly arranged

c. is methylated on specific bases

d. all of the above

Ans: d

22. A ribozyme is an RNA sequence

a. that has Mg2+ ions as a cofactor

b. with catalytic ability to cleave RNA

c. that acts in the spliceosome

d. all of the above

PART B: Testing on the Concepts

8.1 Processing of Eukaryotic Pre-mRNA

23. How is the 5´-Cap added to nascent RNAs?

Ans: A capping enzyme removes the -phosphate from the 5´ end of the nascent RNA emerging from the surface of a RNA polymerase II complex. A separate subunit of the capping enzyme then transfers a GMP moiety from a GTP donor to the 5´-diphosphate of the nascent transcript, creating a 5´-5´-triphosphate structure. Separate enzymes transfer a methyl group from an S-adenosinemethionine donor to the N7 position of the guanine and the 2´ oxygens of riboses at the 5´ end of the nascent RNA.

24. In animal cells, nearly all cytoplasmic mRNAs have a 3´ poly(A) tail, which is added to the pre-mRNA before splicing. What proteins are involved in polyadenylation? Indicate their order of association with pre-mRNA and their functions.

Ans: (1) Poly(A) signal, which often is an AAUAAA sequence and binds the cleavage and polyadenylation specificity factor (CPSF); (2) poly(A) site, at which cleavage occurs and addition of A residues begins; and (3) G/U-rich region, which binds cleavage stimulatory factor (CStF). Polyadenylation of pre-mRNA begins with binding of CPSF, which is composed of several proteins, to the poly(A) signal. Then, at least three other proteins, including CStF, bind to CPSF-RNA complex; interaction of CStF with the downstream GU-rich sequence stabilizes the entire complex. Binding of poly(A) polymerase to the complex then stimulates cleavage of the RNA at the poly(A) site and subsequent addition of A residues. Polymerization of A residues initially occurs slowly but its rate is enhanced by binding of multiple copies of a protein called PABII. The mechanism by which the length of the poly(A) tail is restricted to about 200 nucleotides is not known.

25. What are hnRNP proteins? How were they identified?

Ans: HnRNP proteins are the major protein components of heterogeneous nuclear RNA particles, which consist of unspliced nuclear mRNA and other nuclear RNAs. To identify hnRNP proteins, investigators exposed cells to UV irradiation, which causes covalent cross-links to form between RNA and closely associated proteins. Chromatography of nuclear extracts from irradiated cells on an oligo-dT cellulose column will bind the poly(A) tails of unspliced mRNAs and can be used to recover proteins that have become cross-linked to these RNAs.

26. SnRNP-dependent splicing of pre-mRNA is thought to have evolved from the self-splicing properties inherent in the sequence of either group I or II introns. Alternative splicing of pre-mRNAs processed in spliceosomes has been demonstrated, whereas this phenomenon does not occur in RNA transcripts that undergo selfsplicing. Explain this difference.

Ans: In the case where splicing is self-mediated in response to sequence features, splicing is an intrinsic property of the molecule. This is the case with group I and II introns. In snRNP-mediated splicing, the splicing process, although responsive to pre-mRNA sequence, is not dictated by the sequence of the RNA being spliced. For this reason, splicing of the molecule may be regulated and alternative RNA splicing may occur.

27. The finding that the short consensus sequence at the 5´ end of introns is complementary to a sequence near the 5´ end of U1 snRNA suggested that this snRNA must interact with pre-mRNA for splicing to occur. Describe three types of experimental evidence that indicate U1 snRNA is required for splicing.

Ans: Addition of antiserum specific for U1 snRNP prevents in vitro splicing. A synthetic oligonucleotide with the sequence of the 5´ end of U1 snRNA competes for the normal U1 snRNA and prevents splicing. Mutations in either the 5´ splice site of pre-mRNA or U1 snRNA prevent splicing; however, if a compensatory mutation that restores base pairing is present in the second component, then splicing occurs.

28. The spliceosomal splicing cycle involves ordered interactions among a pre-mRNA and several U snRNPs. According to the current model of spliceosomal splicing, which intermediate(s) in the splicing of a pre-mRNA containing one intron should be immunoprecipitated by anti-U2 snRNP? Which additional intermediate(s) should be immunoprecipitated by anti-U2AF?

Ans: Four different potential intermediates should be immunoprecipitated by anti-U2 snRNP: (1) a structure in the process of joining the two exons together but still containing the intron; (2) a structure that contains the excised intron in lariat form; (3) the pre-mRNA with U2 snRNP bound to the 5´ end of the intron; and (4) a structure consisting of the pre-mRNA, U1 snRNP, and U2 snRNP bound to the branch site. Because U2AF assists U2 snRNP in binding the pre-mRNA, antibodies against this protein will immunoprecipitate the same complexes.

29. In yeast, U2 snRNA base-pairs to a short sequence near branch-point A in introns. In higher eukaryotes, this branch-point sequence is not highly conserved, and a protein called U2AF promotes binding of U2 snRNA to pre-mRNA. You have produced mice with a knockout mutation in the U2AF gene. Would you expect mice heterozygous for the U2AF knockout mutation to be viable? Would you expect mice homozygous for the U2AF knockout mutation to be viable?

Ans: Association of the U2 snRNP with pre-mRNA is a necessary step in splicing. In higher eukaryotes, viability depends on proper splicing of pre-mRNA. However, assuming that U2AF normally is produced in excess, heterozygous knockout mice most likely would have sufficient U2AF to support splicing. Thus little, if any, effect on the viability of these mice would be observed. Because proper splicing of pre-mRNA is a necessity for the viability of higher eukaryotes, a homozygous knockout mutation in mice for UA2AF would be expected to be lethal. Nonlethality would indicate the existence of redundancy in the pre-mRNA splicing mechanism. However, biological systems often exhibit redundancy to protect the organism, so the homozygous knockout mice might survive.

8.2 Regulation of Pre-mRNA Processing

30. Describe how the Sex-lethal (Sxl) protein is regulated during the development of Drosophila females.

Ans: Early in development, females utilize the Pe promoter to synthesize sxl mRNA containing exons 1 and 2, which is spliced normally, resulting in the production of early Sxl protein. Later in development, the Pl promoter located upstream is utilized, producing exons 1 through 4. Sxl protein made earlier binds to this sxl mRNA, preventing splicing of exons 2 and 3. The resulting mRNA, containing only exons 1, 2, and 4, is translated into functional late Sxl protein, which also binds to the late sxl pre-mRNA, ensuring its continued production.

8.4 Cytoplasmic Mechanisms of Post-Transcriptional Control

31. The oocytes of multicellular animals contain stored mRNAs that encode numerous proteins required for early embryonic development. These proteins, however, are not translated until after fertilization and, therefore, a mechanism must be in place to ensure the stored mRNAs remain intact and not translated before they are needed. Discuss the mechanism that keeps these stored mRNAs from being translated in the oocyte.

Ans. Stored mRNAs in oocytes have short poly(A) tails, consisting of ~20–40 residues. These short tails can bind only a few molecules of cytoplasmic poly(A)-binding protein (PABPI), which is not enough to interact with the initiation factor, eIF4G. Following fertilization, the poly(A) tail increases in length with the addition of ≈150 A residues. This facilitates the binding of several PABPI molecules, allowing them to interact with eIF4G in a multimeric complex with eIF4E, other initiation factors, and the cap at the 5´ end of the mRNA. The stable conformation that forms is required for the initiation of translation. Thus stored mRNAs are not translated efficiently because their short poly(A) tails do not provide enough binding sites to allow PABPI to implement the stability the complex requires for translation initiation.

8.5 Processing of rRNA and tRNA

32. The tissue-specific expression of antisense RNA is one experimental approach for selectively shutting down production of a protein. For example, some researchers have proposed that this approach could be used to regulate the production of pollen in tobacco, oilseed rape, and maize. The controlled production of sterile male plants, for example, would eliminate the problem of self-fertilization in the production of hybrid maize seed. In this approach, expression of antisense RNA would be controlled by coupling it to a promoter that is specific to anthers, the part of flowers where pollen is produced. Alternatively, the RNase activity inherent in self-splicing RNA might provide a sequence-specific means to regulate pollen production. Discuss how a catalytic RNA (i.e., a ribozyme) might be designed to prevent the expression of proteins needed for pollen production.

Ans: Some RNAs are capable of both sequence-specific base pairing and catalytic activity as an RNase. For example, when the 400-nucleotide-long intron sequence from Tetrahymena rRNA, a group I self-splicing RNA, is synthesized in a test tube, it folds and can bind two substrates, a guanine nucleotide and a substrate RNA chain. This synthetic intron then catalyzes the covalent attachment of the G to the substrate RNA, thereby cleaving the substrate RNA at a specific site. The release of the two RNA fragments frees the catalytic RNA for repeated rounds of catalysis. In principle, through the inclusion of the appropriate sequence for base pairing, a catalytic RNA can be designed that will bind to any substrate RNA and sever it at a specific site. Engineering a DNA sequence encoding a properly designed catalytic RNA under control of a tissue-specific set of promoter/enhancer elements and incorporating it into the germ line of plants could result in the tissuespecific synthesis of a ribozyme capable of selectively destroying a differentiation-specific mRNA required for pollen production.

33. Guanosine in the form of free guanosine G, GMP, GDP, or GTP functions as a cofactor for self-splicing of Tetrahymena rRNA. Self-splicing exhibits a Km of 32 mM for G and is competitively inhibited by inosine, a nucleoside analog. What do these properties suggest regarding the interaction of G and Tetrahymena rRNA?

Ans: Guanosine does not function as an energy source. If it did, only a high-energy phosphate form such as GTP would be effective in self-splicing. That the reaction is saturable for G (i.e., there are a Vmax and Km) and G can be competed against suggest that there must be a folded domain in Tetrahymena rRNA capable of specifically binding G. In other words, the rRNA has a cofactor-binding site similar to that present in some enzymes.

34. What are the main features of splicing in pre-tRNA that distinguish it from splicing in pre-mRNA?

Ans: Splicing of pre-tRNA does not involve spliceosomes. In the first step, an endonuclease-catalyzed reaction excises the intron, which is released as a linear fragment, and a 2,3-cyclic monophosphate ester forms on the cleaved end of the 5 exon. A multistep reaction that requires the energy derived from hydrolysis of one GTP and one ATP then joins the two exons. In contrast, pre-mRNA splicing occurs in spliceosomes, involves two transesterification reactions, releases the intron as a lariat structure, and does not require GTP. Although these transesterification reactions do not require ATP hydrolysis, it probably is necessary for the rearrangements that occur in the spliceosome.