Transcription is catalyzed by DNA-directed RNA polymerase, a complex enzyme that synthesizes RNA complementary to a segment of one strand (the template strand) of duplex DNA, starting from ribonucleoside 5'-triphosphates. To initiate transcription, RNA polymerase binds to a DNA site called a promoter. Bacterial RNA polymerase requires a special subunit for recognizing the promoter. As the first committed step in transcription, binding of RNA polymerase to promoters is subject to many forms of regulation. Eukaryotic cells have three different types of RNA polymerases. Transcription stops at specific sequences called terminators. Many copies of an RNA chain can be transcribed simultaneously from a single gene.
Ribosomal RNAs and transfer RNAs are made from longer precursor RNAs that are trimmed by nucleases, and some bases are modified enzymatically to yield the mature RNAs. In eukaryotes, messenger RNAs are also formed from longer precursors. Primary RNA transcripts often contain noncoding regions called introns, which are removed by splicing. Group I introns are found in rRNAs and their excision requires a guanosine cofactor. Some group I and some group II introns are capable of self-splicing; no protein enzymes are required. Nuclear mRNA precursors have a third class of introns that are spliced with the aid of RNA-protein complexes called snRNPs. The fourth class of introns, found in some tRNAs, are the only ones known to be spliced by protein enzymes. Messenger RNAs are also modified by addition of a 7-methylguanosine residue at the 5' end, and cleavage and polyadenylation at the 3' end to form a long poly(A) tail.
The self splicing introns and the RNA component of RNase P (the enzyme that cleaves the 5' end of tRNA precursors) form a new class of biological catalysts called ribozymes. These have the properties of true enzymes and are effective catalysts. They promote two types of reaction, hydrolytic cleavage and transesterification, using RNA as substrate. Combinations of these reactions are promoted by the excised group I rRNA intron from Tetrahymena, resulting in a type of RNA polymerization reaction. The study of these reactions and of introns themselves has provided insights into likely pathways for biochemical evolution.
Polynucleotide phosphorylase can reversibly form RNA-like polymers from ribonucleoside 5'diphosphates, adding or removing ribonucleotides at the 3'-hydroxyl end of the polymer. It acts in vivo to degrade RNA.
RNA-directed DNA polymerases, also called reverse transcriptases, are produced in animal cells infected by RNA viruses called retroviruses. These enzymes transcribe the viral RNA into DNA. This process can be used experimentally to form complementary DNA. Many eukaryotic transposons are related to retroviruses, and their mechanism of transposition includes an RNA intermediate. The enzyme that synthesizes telomeres, called telomerase, is a specialized reverse transcriptase that contains an internal RNA template.
RNA-directed RNA polymerases, or replicases, are found in bacterial cells infected with certain RNA viruses. They are template-specific for the viral RNA.
The existence of catalytic RNAs and pathways for the interconversion of RNA and DNA has led to speculation that the earliest living things were made up entirely or largely of RNA molecules that served both for information storage and for catalysis of replication.
General
Darnell, J.E., Jr. (1985) RNA. Sci. Am. 253 (October), 68-78.
Evolution of Catalytic Function. (1987) Cold Spring Harb. Symp. Quant. Biol. 52.
An excellent source for articles on catalytic RNA, evolution, and many other topics discussed in this chapter.
Jacob, F. & Monod, J. (1961) Genetic regulatory mechanisms in the synthesis of proteins. J. Mol. Biol. 3, 318-356.
A classic article that introduced many important ideas.
Watson, J.D., Hopkins, N.H., Roberts, J.W., Steitz, J.A., & Weiner, A.M. (1987) Molecular Biology of the Gene, 4th edn, The Benjamin/ Cummings Publishing Company, Menlo Park, CA.
DNA-Directed RNA Synthesis
Conaway, J.W. & Conaway, R.C. (1991) Initiation of eukaryotic messenger RNA synthesis. J. Biol. Chem. 266, 17721-17724.
A good minireview.
McClure, W.R. (1985) Mechanism and control of transcription initiation in prokaryotes. Annu. Rev. Biochem. 54, 171-204.
Platt, T. (1986) l~anscription termination and the regulation of gene expression. Annu. Rev. Biochem. 55, 339-372.
Sawadogo, M. & Sentenac, A. (1990) RNA polymerase B (II) and general transcription factors. Annu. Rev. Biochem. 59, 711-754.
A good review of eukaryotic RNA polymerase 11.
RNA Processing
Breitbart, R.E., Andreadis, A., & Nadal-Ginard, B. (1987) Alternative splicing: a ubiquitous mechanism for the generation of multiple protein isoforms from single genes. Annu. Rev. Biochem. 56, 467-495.
Cech, T.R. (1986) RNA as an enzyme. Sci. Am. 255 (November), 64-75.
Cech, T.R. (1987) The chemistry of self splicing RNA and RNA enzymes. Science 236, 1532-1539.
Deutscher, M.P. (1990) Ribonucleases, tRNA nucleotidyltransferase, and the 3' processing of tRNA. Prog. Nucleic Acid Res. Mol. Biol. 39, 209240.
A good overview of tRNA processing reactions.
Green, M.R. (1986) Pre-mRNA splicing. Annu. Rev. Genet. 20, 671-708.
McCorkle, G.M. & Altman, S. (1987) RNAs as catalysts: a new class of enzyme. J. Chem. Educ. 64, 221-226.
Pace, N.R. & Smith, D. t1990) Ribonuclease P: function and variation. J. Biol. Chem. 265, 35873590.
Ross, J. (1989) The turnover of messenger RNA. Sci. Am. 260 (April), 48-55.
Sharp, P.A. (1987) Splicing of messenger RNA precursors. Science 235, 766-771.
Wahle, E. & Keller, W. (1992) The biochemistry of 3'-end cleavage and polyadenylation of messenger RNA precursors. Annu. Rev. Biochem. 61, 419-440. Wickens, M. (1990) How the messenger got its tail: addition of poly(A) in the nucleus. Trends Biochem. ~Sci. 15, 277-281.
RNA-Directed RNA or DNA Synthesis
Belfort, M. (1991) Self splicing introns in prokaryotes: migrant fossils? Cell 64, 9-11.
A discussion of the evolutionary significance of prokaryotic introns.
Bishop, J.M. (1991) Molecular themes in oncogenesis. Cell 64, 235-248.
A good overuiew of oncogenes; it introduces a series of more detailed reuiews included in the same issue of Cell.
Blackburn, E.H. (1991) Telomeres. Trends Biochem. Sci. 16, 378-381.
Blackburn, E.H. (1992) Telomerases. Annu. Rev. Biochem. 61, 113-129.
Boeke, J.D. (1990) Reverse transcriptase, the end of the chromosome, and the end of life. Cell 61, 193195.
The possible role of telomerase in regulating the life span of an organism.
Dorit, R.L., Schoenbach, L., & Gilbert, W. (1990) How big is the universe of exons? Science 250, 1377-1382.
Interesting speculation on the origin and function of exons.
Gallo, R.C. & Montagnier, L. (1988) AIDS in 1988. Sci. Am. 259 (October), 40-48.
The introductory article to an entire Scientific American issue deuoted to AIDS.
Kingsman, A.J. & Kingsman, S.M. (1988) Ty: a retroelement moving forward. Cell 53, 333-335. Describes a well-studied yeast transposon related to retrouiruses.
Pace, N.R. (1991) Origin of life-facing up to the physical setting. Cell 65, 531-533.
A discussion of the conditions believed to have existed when Life began euolving.
Perlman, P.S. & Butow, R.A. (1989) Mobile introns and intron-encoded proteins. Science 246, 1106-1109.
This article describes a special class of introns capable of colonizing homologous genes that lack introns.
Temin, H.M. (1976) The DNA provirus hypothesis: the establishment and implications of RNAdirected DNA synthesis. Science 192, 1075-1080. A discussion of the original proposal for reuerse transcription in retrouiruses.
Varmus, H. (1987) Reverse transcription. Sci. Am. 257 (September), 56-64.
Varmus, H.E. (1989) Reverse transcription in bacteria. Cell 56, 721-724.
1. RNA Polymerase How long would it take for the E. coli RNA polymerase to synthesize the primary transcript for E. coli rRNAs (6500 bases)?
2. Error Correction by RNA Polymerases DNA polymerases are capable of editing and error correction, but RNA polymerases do not appear to have this capacity. Given that a single base error in either replication or transcription can lead to an error in protein synthesis, can you give a possible biological explanation for this striking difference? 3. The Rate of Transcription From what you know of the rate at which E. coli RNA polymerase synthesizes RNA, predict how far the transcription "bubble" formed by RNA polymerase will move along the DNA in 10 s.
4. RNA Posttranscriptional Processing Predict the likely effects of a mutation in the sequence (5')AAUAAA in a eukaryotic mRNA transcript.
5. Coding vs. Template Strcznds The RNA genome of phage Qβ is the nontemplate or (+) strand, and when introduced into the cell it functions as an mRNA. Suppose the RNA replicase of phage Qβ synthesized primarily (-) strand RNA and uniquely incorporated it into the virus particles, rather than (+) strands. What would be the fate of the (-) strands when they entered a new cell?
What enzyme would such a ( - ) strand virus need to include in the virus particle to successfully invade a host cell?
6. The Chemistry of Nucleic Acid Biosynthesis Describe three properties common to the reactians catalyzed by DNA polymerase, RNA polymerase, reverse transcriptase, and RNA replicase.
7. RNA Splicing What is the minimum number of transesterification reactions needed to splice an intron from an mRNA transcript? Why?
8. Telomerase Assuming that the RNA component of telomerase is fixed within the protein structure, in what respect might the active site of this enzyme differ from the active site of reverse transcriptases, RNA polymerases, and DNA polymerases? (Hint: The latter three enzymes add one nucleotide at a time. )
9. RNA Genomes The RNA viruses have relatively small genomes. For example, the singlestranded RNAs of retroviruses have about 10,000 nucleotides and the Qβ RNA is only 4,220 nucleotides long. Given the properties of reverse transcriptase and RNA replicase described in this chapter, can you suggest a reason for the small size of these viral genomes?
