Berg J.M., Tymoczko J.L., Stryer L. Biochemistry
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III. Synthesizing the Molecules of Life 28. RNA Synthesis and Splicing 28.4. The Discovery of Catalytic RNA Was Revealing in Regard to Both Mechanism and Evolution
Figure 28.36. Self-Splicing Mechanism. The catalytic mechanism of the selfsplicing intron from Tetrahymena includes
a series of transesterification reactions. [After T. Cech. RNA as an enzyme. Copyright © 1986 by Scientific American,
Inc. All rights reserved.]
III. Synthesizing the Molecules of Life 28. RNA Synthesis and Splicing 28.4. The Discovery of Catalytic RNA Was Revealing in Regard to Both Mechanism and Evolution
Figure 28.37. Comparison of Splicing Pathways. The exons being joined are shown in blue and yellow and the
attacking unit is shown in green. The catalytic site is formed by the intron itself (red) in group I and group II splicing. In
contrast, the splicing of nuclear mRNA precursors is catalyzed by snRNAs and their associated proteins in the
spliceosome. [After P. A. Sharp. Science 235(1987):769.]
III. Synthesizing the Molecules of Life 28. RNA Synthesis and Splicing
Summary
Transcription Is Catalyzed by RNA Polymerase
All cellular RNA molecules are synthesized by RNA polymerases according to instructions given by DNA templates.
The activated monomer substrates are ribonucleoside triphosphates. The direction of RNA synthesis is 5
3 , as in
DNA synthesis. RNA polymerases, unlike DNA polymerases, do not need a primer and do not possess proofreading
nuclease activity.
RNA polymerase in E. coli is a multisubunit enzyme. The subunit composition of the ~500-kd holoenzyme is α
2
β β σ
and that of the core enzyme is α
2
β β . Transcription is initiated at promoter sites consisting of two sequences, one
centered near -10 and the other near -35; that is, 10 and 35 nucleotides away from the start site in the 5
(upstream)
direction. The consensus sequence of the -10 region is TATAAT. The σ subunit enables the holoenzyme to recognize
promoter sites. When the growth temperature is raised, E. coli expresses a special σ that selectively binds the distinctive
promoter of heat-shock genes. RNA polymerase must unwind the template double helix for transcription to take place.
Unwinding exposes some 17 bases on the template strand and sets the stage for the formation of the first phosphodiester
bond. RNA chains usually start with pppG or pppA. The σ subunit dissociates from the holoenzyme after the initiation of
the new chain. Elongation takes place at transcription bubbles that move along the DNA template at a rate of about 50
nucleotides per second. The nascent RNA chain contains stop signals that end transcription. One stop signal is an RNA
hairpin, which is followed by several U residues. A different stop signal is read by the rho protein, an ATPase. In E. coli,
precursors of transfer RNA and ribosomal RNA are cleaved and chemically modified after transcription, whereas mRNA
is used unchanged as a template for protein synthesis.
Eukaryotic Transcription and Translation Are Separated in Space and Time
RNA synthesis in eukaryotes takes place in the nucleus, whereas protein synthesis takes place in the cytoplasm. There
are three types of RNA polymerase in the nucleus: RNA polymerase I makes ribosomal RNA precursors, II makes
messenger RNA precursors, and III makes transfer RNA precursors. Eukaryotic promoters are complex, being composed
of several different elements. Promoters for RNA polymerase II are located on the 5
side of the start site for
transcription. Each consists of a TATA box centered between -30 and -100 and additional upstream sequences. They are
recognized by proteins called transcription factors rather than by RNA polymerase II. The saddle-shaped TATA-box-
binding protein unwinds and sharply bends DNA at TATA-box sequences and serves as a focal point for the assembly of
transcription complexes. The TATA-box-binding protein initiates the assembly of the active transcription complex. The
activity of many promoters is greatly increased by enhancer sequences that have no promoter activity of their own.
Enhancer sequences can act over distances of several kilobases, and they can be located either upstream or downstream
of a gene.
The Transcription Products of All Three Eukaryotic Polymerases Are Processed
The 5
ends of mRNA precursors become capped and methylated in the course of transcription. A 3 poly(A) tail is added
to most mRNA precursors after the nascent chain has been cleaved by an endonuclease. RNA editing processes alter the
nucleotide sequence of some mRNAs, such as the one for apolipoprotein B.
The splicing of mRNA precursors is carried out by spliceosomes, which consist of small nuclear ribonucleoprotein
particles (snRNPs). Splice sites in mRNA precursors are specified by sequences at ends of introns and by branch sites
near their 3
ends. The 2 -OH group of an adenosine residue in the branch site attacks the 5 splice site to form a lariat
intermediate. The newly generated 3
-OH terminus of the upstream exon then attacks the 3 splice site to become joined
to the downstream exon. Splicing thus consists of two transesterification reactions, with the number of phosphodiester
bonds remaining constant during reactions. Small nuclear RNAs in spliceosomes catalyze the splicing of mRNA
precursors. In particular, U2 and U6 snRNAs form the active centers of spliceosomes.
The Discovery of Catalytic RNA Was Revealing with Regard to Both Mechanism And
Evolution
Some RNA molecules, such as the 26S ribosomal RNA precursor from Tetrahymena, undergo self-splicing in the
absence of protein. A self-modified version of this rRNA intron displays true catalytic activity. Spliceosome-catalyzed
splicing may have evolved from self-splicing. The discovery of catalytic RNA has opened new vistas in our exploration
of early stages of molecular evolution.
Key Terms
transcription
RNA polymerase
promoter sites
transcription factor
footprinting
consensus sequence
sigma subunit
transcription bubble
rho (ρ) protein
TATA box
enhancer
pre-mRNA
5
cap
poly(A) tail
RNA editing
RNA splicing
spliceosome
small nuclear RNA (snRNA)
alternative splicing
catalytic RNA
self-splicing
III. Synthesizing the Molecules of Life 28. RNA Synthesis and Splicing
Problems
1.
Complements. The sequence of part of an mRNA is
What is the sequence of the DNA coding strand? Of the DNA template strand?
See answer
2.
Checking for errors. Why is RNA synthesis not as carefully monitored for errors as is DNA synthesis?
See answer
3.
Speed is not of the essence. Why is it advantageous for DNA synthesis to be more rapid than RNA synthesis?
See answer
4.
Potent inhibitor. Heparin inhibits transcription by binding to RNA polymerase. What properties of heparin allow it
to bind so effectively to RNA polymerase?
See answer
5.
A loose cannon. Sigma protein by itself does not bind to promoter sites. Predict the effect of a mutation enabling σ to
bind to the -10 region in the absence of other subunits of RNA polymerase.
See answer
6.
Stuck sigma. What would be the likely effect of a mutation that would prevent σ from dissociating from the RNA
polymerase core?
See answer
7.
Transcription time. What is the minimum length of time required for the synthesis by E. coli polymerase of an
mRNA encoding a 100-kd protein?
See answer
8.
Between bubbles. How far apart are transcription bubbles on E. coli genes that are being transcribed at a maximal
rate?
See answer
9.
A revealing bubble. Consider the synthetic RNA-DNA trancription bubble illustrated here. Let us refer to the coding
DNA strand, the template strand, and the RNA strand as strands 1, 2, and 3, respectively.
(a) Suppose that strand 3 is labeled with
32
P at its 5 end and that polyacrylamide gel electrophoresis is carried out
under nondenaturing conditions. Predict the autoradiographic pattern for (i) strand 3 alone, (ii) strands 1 and 3, (iii)
strands 2 and 3, (iv) strands 1, 2, and 3, and (v) strands 1, 2, and 3 and core RNA polymerase.
(b) What is the likely effect of rifampicin on RNA synthesis in this system?
(c) Heparin blocks elongation of the RNA primer if it is added to core RNA polymerase before the onset of
transcription but not if added after transcription starts. Account for this difference.
(d) Suppose that synthesis is carried out in the presence of ATP, CTP, and UTP. Compare the length of the longest
product obtained with that expected when all four ribonucleoside triphosphates are present.
See answer
10.
Abortive cycling. Di- and trinucleotides are occasionally released from RNA polymerase at the very start of
transcription, a process called abortive cycling. This process requires the restart of transcription. Suggest a plausible
explanation for abortive cycling.
See answer
11.
Polymerase inhibition. Cordycepin inhibits poly(A) synthesis at low concentrations and RNA synthesis at higher
concentrations.
(a) What is the basis of inhibition by cordycepin?
(b) Why is poly(A) synthesis more sensitive to the presence of cordycepin?
(c) Does cordycepin need to be modified to exert its effect?
See answer
12.
An extra piece. What is the amino acid sequence of the extra segment of protein synthesized in a thalassemic
patient having a mutation leading to aberrant splicing (see Figure 28.28)? The reading frame after the splice site
begins with TCT.
See answer
13.
A long-tailed messenger. Another thalassemic patient had a mutation leading to the production of an mRNA for the
β chain of hemoglobin that was 900 nucleotides longer than the normal one. The poly(A) tail of this mutant mRNA
was located a few nucleotides after the only AAUAAA sequence in the additional sequence. Propose a mutation
that would lead to the production of this altered mRNA.
See answer
Mechanism Problem
14.
RNA editing. Many uridine molecules are inserted into some mitochondrial mRNAs in trypanosomes. The uridine
residues come from the poly(U) tail of a donor strand. Nucleoside triphosphates do not participate in this reaction.
Propose a reaction mechanism that accounts for these findings. (Hint: Relate RNA editing to RNA splicing.)
See answer
Chapter Integration Problems
15.
Proteome complexity. What processes considered in this chapter make the proteome more complex than the
genome? What processes might further enhance this complexity?
See answer
16.
Separation technique. Suggest a means by which you could separate mRNA from the other types of RNA in a
eukaryotic cell.
See answer
Data Interpretation Problems
17.
Run-off experiment. Nuclei were isolated from brain, liver, and muscle. The nuclei were then incubated with α-
32
P-
UTP under conditions that allow RNA synthesis, except that an inhibitor of RNA initiation was present. The
radioactive RNA was isolated and annealed to various DNA sequences that had been attached to a gene chip. In the
adjoining graphs, the intensity of the shading indicates roughly how much mRNA was attached to each RNA
sequence.
(a) Why does the intensity of hybridization differ between genes?
(b) What is the significance of the fact that some of the RNA molecules display different hybridization patterns in
different tissues?
(c) Some genes are expressed in all three tissues. What would you guess is the nature of these genes?
(d) Suggest a reason why an initiation inhibitor was included in the reaction mixture.
See answer
18.
Christmas trees. The adjoining autoradiograph depicts several bacterial genes undergoing transcription. Identify the
DNA. What are the strands of increasing length? Where is the beginning of transcription? The end of transcription?
On the page, what is the direction of RNA synthesis? What can you conclude about the number of enzymes
participating in RNA synthesis on a given gene?
See answer
III. Synthesizing the Molecules of Life 28. RNA Synthesis and Splicing
Selected Readings
Where to begin
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Biol. 63: 311-317. (PubMed)
R. Losick. 1998. Summary: Three decades after sigma Cold Spring Harbor Symp. Quant. Biol. 63: 653-666. (PubMed)
J.E. Darnell Jr.. 1985. RNA Sci. Am. 253: (4) 68-78. (PubMed)
T.R. Cech. 1986. RNA as an enzyme Sci. Am. 255: (5) 64-75. (PubMed)
P.A. Sharp. 1994. Split genes and RNA splicing Cell 77: 805- 815. (PubMed)
T.R. Cech. 1990. Self-splicing and enzymatic activity of an intervening sequence RNA from Tetrahymena Biosci. Rep.
10: 239-261. (PubMed)
C. Guthrie. 1991. Messenger RNA splicing in yeast: Clues to why the spliceosome is a ribonucleoprotein Science 253:
157-163. (PubMed)
Books
Lewin, B., 2000. Genes (7th ed.). Oxford University Press.
Kornberg, A., and Baker, T. A., 1992. DNA Replication (2d ed.). W. H. Freeman and Company.
Lodish, H., Berk, A., Zipursky, S. L., Matsudaira, P., Baltimore, D., and Darnell, J., 2000. Molecular Cell Biology (4th
ed.). W. H. Freeman and Company.
Watson, J. D., Hopkins, N. H., Roberts, J. W., Steitz, J. A., and Weiner, A. M., 1987. Molecular Biology of the Gene (4th
ed.). Benjamin Cummings.
Gesteland, R. F., Cech, T., and Atkins, J. F. (Eds.), 1999. The RNA World (2d ed.). Cold Spring Harbor Laboratory
Press.
RNA polymerases
P. Cramer, D.A. Bushnell, and R.D. Kornberg. 2001. Structural basis of transcription: RNA polymerase II at 2.8 Å
resolution Science 292: 1863-1875. (PubMed)
A.L. Gnatt, P. Cramer, J. Fu, D.A. Bushnell, and R.D. Kornberg. 2001. Structural basis of transcription: An RNA
polymerase II elongation complex at 3.3 Å resolution Science 292: 1876-1882. (PubMed)
G. Zhang, E.A. Campbell, L. Minakhin, C. Richter, K. Severinov, and S.A. Darst. 1999. Crystal structure of Thermus
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E.A. Campbell, N. Korzheva, A. Mustaev, K. Murakami, S. Nair, A. Goldfarb, and S.A. Darst. 2001. Structural
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G.M. Cheetham and T.A. Steitz. 1999. Structure of a transcribing T7 RNA polymerase initiation complex Science 286:
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Initiation and elongation
S. Buratowski. 2000. Snapshots of RNA polymerase II transcription initiation Curr. Opin. Cell Biol. 12: 320-325.
(PubMed)
J.W. Conaway and R.C. Conaway. 1999. Transcription elongation and human disease Annu. Rev. Biochem. 68: 301-319.
(PubMed)
J.W. Conaway, A. Shilatifard, A. Dvir, and R.C. Conaway. 2000. Control of elongation by RNA polymerase II Trends
Biochem. Sci. 25: 375-380. (PubMed)
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D. Reines, R.C. Conaway, and J.W. Conaway. 1999. Mechanism and regulation of transcriptional elongation by RNA
polymerase II Curr. Opin. Cell Biol. 11: 342-346. (PubMed)
Promoters, enhancers, and transcription factors
M. Merika and D. Thanos. 2001. Enhanceosomes Curr. Opin. Genet. Dev. 11: 205-208. (PubMed)
J.M. Park, B.S. Gim, J.M. Kim, J.H. Yoon, H.S. Kim, J.G. Kang, and Y.J. Kim. 2001. Drosophila mediator complex is
broadly utilized by diverse gene-specific transcription factors at different types of core promoters Mol. Cell. Biol. 21:
2312-2323. (PubMed) (Full Text in PMC)
S. Fiering, E. Whitelaw, and D.I. Martin. 2000. To be or not to be active: The stochastic nature of enhancer action
Bioessays 22: 381-387. (PubMed)
M. Hampsey and D. Reinberg. 1999. RNA polymerase II as a control panel for multiple coactivator complexes Curr.
Opin. Genet. Dev. 9: 132-139. (PubMed)
L. Chen. 1999. Combinatorial gene regulation by eukaryotic transcription factors Curr. Opin. Struct. Biol. 9: 48-55.
(PubMed)
C.W. Muller. 2001. Transcription factors: Global and detailed views Curr. Opin. Struct. Biol. 11: 26-32. (PubMed)
H. Sakurai and T. Fukasawa. 2000. Functional connections between mediator components and general transcription
factors of Saccharomyces cerevisiae J. Biol. Chem. 275: 37251-37256. (PubMed)
P. Droge and B. Muller-Hill. 2001. High local protein concentrations at promoters: Strategies in prokaryotic and
eukaryotic cells Bioessays 23: 179-183. (PubMed)
J.W. Fickett and A.G. Hatzigeorgiou. 1997. Eukaryotic promoter recognition Genome Res. 7: 861-878. (PubMed)
S.T. Smale, A. Jain, J. Kaufmann, K.H. Emami, K. Lo, and I.P. Garraway. 1998. The initiator element: A paradigm for
core promoter heterogeneity within metazoan protein-coding genes Cold Spring Harbor Symp. Quant. Biol. 63: 21-31.
(PubMed)
Y. Kim, J.H. Geiger, S. Hahn, and P.B. Sigler. 1993. Crystal structure of a yeast TBP/TATA-box complex Nature 365:
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J.L. Kim, D.B. Nikolov, and S.K. Burley. 1993. Co-crystal structure of TBP recognizing the minor groove of a TATA
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R.J. White and S.P. Jackson. 1992. The TATA-binding protein: A central role in transcription by RNA polymerases I, II
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Termination
B.R. Burgess and J.P. Richardson. 2001. RNA passes through the hole of the protein hexamer in the complex with
Escherichia coli Rho factor J. Biol. Chem. 276: 4182-4189. (PubMed)
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three sites Bio-chemistry 40: 2276-2281. (PubMed)
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5 -Cap formation and polyadenylation
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