11RNA Pr o cessing, Nuclear Tr anspor t, and Post -Tr anscr iptional Contr ol
A guide to parallel questions in Working with Molecular Cell Biology: A Study Companion can be found at the end of this chapter.
PART A: Linking Concepts and Facts
Circle the letters corresponding to the appropriate terms/phrases that complete items 1–20.
11.1 Transcription Termination
1.
Termination of transcription in prokaryotes can be dependent on:
a. intron/exon boundary sequences
b. rho factor
c. the 5’UTR
d. all of the above
2. In E. coli, attenuation and antitermination utilize which structure?
a. stem loop structures in RNA
b. stem loop structures in DNA
c. RNA/DNA hybrids
d. differential protein folding
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
11.2 Processing of Eukaryotic mRNA
4. Histone mRNAs lack:
a. poly A tails
b. introns
c. a 3’UTR
d. all of the above
5. 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
6. Which process involves two transesterification reactions?
a. splicing
b. RNA editing
c. capping
d. nuclear transport
7. Splicing joins:
a. two intron sequences
b. two polypeptides
c. two DNA molecules
d. two exon sequences
8. The branch point A residue involved in lariat formation is part of the:
a. intron
b. exon
c. 5’UTR
d. 3’UTR
9. 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
11.3 Regulation of mRNA Processing
10. Sex lethal protein in Drosophila can best be described as a:
a. splicing regulatory factor
b. RNA editing factor
c. transcription factor
d. all of the above
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
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11 RNA Processing, Nuclear Transport, and Post-Transcriptional Control
d. various slo mRNAs with new nonsense codons
11.4 Signal-Mediated Transport through Nuclear Pore Complexes
12. The nuclear pore complex allows for:
a. passive diffusion of smaller molecules
b. import of proteins
c. active transport of very large molecules
d. all of the above
13. Which event does not occur within the nucleus?
a. RNA editing in mammals
b. RNA capping
c. polyadenylation
d. RNA editing in protozoans
14. Transport of unspliced HIV mRNA from the nucleus to the cytoplasm of host cells is promoted by a virus-encoded protein named:
a. Tat
b. Rev
c. nucleoplasmin
d. Ran
15. Which type of RNA participates in nuclear export of mRNA?
a. snRNA
b. hnRNA
c. tRNA
d. rRNA
11.5 Other Mechanisms of Post-Transcriptional Control
16. 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
17. RNA editing is:
a. posttranscriptional alteration of sequences in mRNAs
b. pretranscriptional alteration of sequences in RNAs
c. posttranscriptional joining of two RNA molecules
d. none of the above
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
11.6 Processing of rRNA and tRNA
19. Synthesis of pre-rRNA occurs in the:
a. nucleolus
b. endoplasmic reticulum
c. extranucleolar area of the nucleus
d. cytosol
20. 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
PART B: Understanding the Concepts
11.1 Transcription Termination
21. Briefly describe the molecular mechanism of antitermination. How does it resemble and differ from the molecular mechanism of
attenuation?
22. Mutation of the -phage PL promoter produces a “weak” promoter, which supports minimal transcription of the gene-encoding N
E. coli and on the life cycle of
the virus?
23. How do cell cycle components influence antitermination by HIV?
11.2 Processing of Eukaryotic mRNA
24. How is the 5’-Cap added to nascent RNAs?
11 RNA Processing, Nuclear Transport, and Post-Transcriptional Control
25.
26.
27.
28.
29.
11.3
30.
31.
11.5
32.
11.6
33.
34.
35.
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-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.
What are hnRNP proteins and how were they identified?
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 self-splicing. Explain this difference.
The finding that the short consensus sequence at the
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.
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?
Regulation of mRNA Processing
Describe how the Sex-lethal (Sxl) protein is regulated during the development of Drosophila females.
How does autoregulation of U1A protein production occur?
Other Mechanisms of Post-Transcriptional Control
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 a 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.
Processing of rRNA and tRNA
In yeast, U2 snRNA base-pairs to a short sequence near the 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 homozygous for the U2AF knockout mutation to be viable?
One of the major objectives of the human genome sequencing project is to identify all protein-coding genes. Analysis of the immense
amount of sequence data generated will require excellent computer software. You have been asked to provide computer programmers
with guidelines they can use in developing algorithms for identification of protein-coding genes. Discuss the specific rules that these
algorithms must incorporate in order to identify all protein-coding genes yet avoid identifying spurious protein-coding genes.
Guanosine in the form of free guanosine (G), GMP, GDP, or GTP functions as a cofactor for self-splicing of Tetrahymena rRNA. Selfsplicing 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?
PART C: Answers
1. b; 2. a; 3. d; 4. a; 5. b; 6. a; 7. d; 8. a; 9. d; 10. a; 11. a; 12. d; 13. d ; 14. b; 15. b; 16. b; 17. a; 18. d; 19. a; 20. c
21. Antitermination prevents premature termination of elongation, while attenuation causes premature termination of transcript
elongation. In order for antitermination to occur in -phage and HIV transcription, a virus-encoded protein—
—
interacts with a stem-loop structure in the nascent viral transcript, called nut
-cell proteins then
interact with RNA polymerase and with N and Tat to form a large complex that acts to prevent transcription termination by looping
of the growing mRNA. Thus antitermination involves stem-loop structures and RNA-binding proteins.
22. Since the N protein is an antiterminator for transcription, a defect in N protein would lead to short RNA transcripts. Hence,
important
viral production.
23. In HIV antitermination, the HIV Tat protein and cyclin T bind to the TAR RNA stem-loop structure. Interaction of cyclin T with Cdk 9
allows for phosphorylation of the CTD of RNA polymerase II. These steps promote antitermination. If the cell cycle components cyclin
T and Cdk 9 are not available or inactive, then antitermination cannot occur.
24. 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.
25. (I) Poly-A signal, which often is an AAUAAA sequence and binds the cleavage-and-polyadenylation specificity factor (CPSF). (II) PolyA site, at which cleavage occurs and addition of A residues begins. (III) 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.
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11 RNA Processing, Nuclear Transport, and Post-Transcriptional Control
26. 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.
27. 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.
28. Addition of antiserum specific for U1 snRNP prevents in vitro splicing. A synthetic oligonucleotide with the sequence of the
U1 snRNA competes for the normal U1 snRNA and prevents splici
-mRNA or U1 snRNA
prevent splicing; however, if a compensatory mutation that restores base pairing is present in the second component, then splicing
occurs.
29. 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
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.
30. 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 which prevents 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.
31. U1A protein bind to a 7 –base sequence in the snRNA component of U1 snRNP. This binding site is just upstream of the polyadenylation
site, and can inhibit the polyadenylation reaction. The result is that when the level of U1A exceeds that of U1 snRNA, the excess U1A
binds to its own mRNA and inhibits polyadenylation, resulting in degradation of this U1A RNA. In this way, production of U1A protein
is controlled by the needs of the splicing machinery.
32. Some RNAs are capable of both sequence-specific base pairing and catalytic activity as an RNase. For example, when the 400nucleotide-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 tissue-specific synthesis of a ribozyme capable of selectively destroying a
differentiation-specific mRNA required for pollen production.
33. 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.
34. Since all eukaryotic protein-coding genes are transcribed by RNA polymerase II, the programmers must develop a set of algorithms
capable of recognizing RNA Pol II transcription units. The problem can be simplified by initially considering only the DNA strand that
is transcribed and by specifying sequence features of this strand—upstream and downstream from the start site—that are present in
some or all protein-coding genes. Among the sequence features unique to protein-coding genes are various elements that control
transcription initiation. For example, most Pol II genes that are transcribed at a high rate have a TATA box about 30 bases upstream
from the start site or an initiator element. The TATA box sequence is highly conserved, whereas initiator promoter elements are not.
In contrast, genes with low transcription rates commonly have a GC-rich region (CCAAT or GGGCG) about 100–200 bases upstream of
the start site. The algorithm should include subroutines to recognize these sequence features first as nucleotide clusters and then to
ask if they are located the appropriate distance from a protein-coding region. Because promoter features are not universal, they can
be a helpful, but not sufficient, criterion for recognizing Pol II transcription units. Putative protein-coding regions (open reading
frames) can be recognized as continuous long stretches of anticodons devoid of stop anticodons. The algorithm must include provisions
for recognizing anticodons in any one of the three possible reading frames. As these open reading frames may be interrupted by
introns, the algorithm must be capable of recognizing introns and intron boundaries. Because introns typically do not encode protein,
they usually do not contain many anticodons. There should be a CA at the 5
-pairs with U1 snRNA. Introns should also have sequences
corresponding to U2- and U5-binding sites; these sequences, however, are poorly conserved. Dinucleotide sequences may be too short
to be a useful identification feature. Protein-coding genes also include a downstream sequence (TTATTT or less commonly TAATT)
that codes for the poly-A addition signal (AAUAAA or AUUAA) in primary transcripts. In addition a CA-rich region that codes for GU
in the RNA transcript is expected about 60–85 nucleotides downstream of the poly-A addition signal. The algorithm must include a
subroutine for the recognition of these sequences. Lastly, because any DNA sequence isolated from the human genome may be from
11 RNA Processing, Nuclear Transport, and Post-Transcriptional Control
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either of the two strands, the actual strand sequenced may not be the strand that is transcribed. Thus the algorithms must include
subroutines for recognition of the complementary sequence features described above.
35. Guanosine does not function as an energy source. If it did, only a high-energy phosphate form such as GTP would be effective in selfsplicing. That the reaction is saturable for G (i.e., there is 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.
PART D: Guide to Parallel Questions in Working with Molecular Cell Biology: A Study Companion
Test Bank
Working with
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