Transcription by RNA Polymerases I and III

Transcription by RNA Polymerases I and III Elongation, Modification, and Termination of Transcription... 

Much of the DNA that is transcribed gives rise to mRNA that is translated into protein. However, the most abundant species of RNA are of other types that are not subsequently translated into protein. They are ribosomal RNA (rRNA) and transfer RNA (tRNA) that participate in mRNA translation they are formed in a high rate of transcription of a relatively small number of genes (called rRNA and tRNA genes) by RNA polymerases I and III. 

Geiduschek, E.P. and Kassavetis, G.A. (1995) Comparing transcriptional initiation by RNA polymerase I and RNA polymerase III. Curr. Opin. Cell Biol. 7, 344-351. 

Functional RNA polymerase I and III are also present in the one-cell embyro (Nothias et al., 1996). Injection of a chloramphenical acetyl transferase reporter gene under the control of the RNA polymerase I-dependent ribosomal DNA promoter into the male pronucleus of S phase-arrested, one-cell embryos (the embryos were incubated in the presence of aphidicolin, which inhibits DNA polymerases a and 8) revealed accumulation of the appropriate transcript by G2 of the one-cell embryo. The amount of this transcript was about 20% of that maximally accumulated when the cleavage-arrested embryos were cultured to a time that corresponded chronologically to the two-cell stage and then were analyzed for expression. A similar result was obtained when the S phase-arrested, one-cell embryos were injected with a plasmid bearing the RNA polymerase Ill-dependent adenovirus VAl RNA gene. In this case, the amount of transcript accumulated by G2 of the S phase-arrested, one-cell embryo was around 30% of that maximally accumulated. 

Week and Wagner (1978) compared transcription of isolated nuclei from VSV-infected and uninfected MPC-11 cells to obtain a more direct measurement of nuclear polymerase activity by the method of Smith and Huang (1976). At 2 hr postinfection, they found approximately a 50% decline in the rate of RNA synthesis in vitro by nuclei isolated from infected cells. The toxin a-amanatin was used at a concentration of 1 xg/ml to distinguish the RNA polymerase II activity from that of the more resistant RNA polymerases I and III. During the first hour after infection, there was a rapid loss in the activities of all three RNA polymerases. Subsequently, the level of RNA polymerase II activity continued to decline until 4 hr postinfection while the level of combined RNA polymerase I and III activity remained constant. 

Nuclear rRNA precursors and cytoplasmic rRNA formed by RNA polymerase I account for some 70+% of mammalian cell RNA and 35-40% of total RNA synthesis. The products of RNA polymerase II, hnRNA and mRNA, comprise some 10% of the total RNA and 55-60% of total synthesis. The hnRNA and mRNA transcripts are unstable and have half-lives in higher eukaryotes of about 24 h, compared with an average half-life in bacteria of 90 s. Pre-tRNAs, synthesized along with 5 S rRNA by RNA polymerase III, are relatively stable, with an average half-life of 4-6 days. 

Rameau, G., Puglia, K., Crowe, A., Sethy, I., and Willis, I. (1994). A mutation in the second largest subunit of TFIIIC increases a rate limiting step in transcription by RNA polymerase III. Mol. Cell. Biol. 14, 822-830. 

Transcription is catalyzed by DNA-dependent RNA polymerases. These act in a similar way to DNA polymerases (see p. 240), except that they incorporate ribonucleotides instead of deoxyribonucleotides into the newly synthesized strand also, they do not require a primer. Eukaryotic cells contain at least three different types of RNA polymerase. RNA polymerase I synthesizes an RNA with a sedimentation coef cient (see p. 200) of 45 S, which serves as precursor for three ribosomal RNAs. The products of RNA polymerase II are hnRNAs, from which mRNAs later develop, as well as precursors for snRNAs. Finally, RNA polymerase III transcribes genes that code for tRNAs, 5S rRNA, and certain snRNAs. These precursors give rise to functional RNA molecules by a process called RNA maturation (see p. 246). Polymerases II and III are inhibited by a-amanitin, a toxin in the Amanita phalloides mushroom. 

In eukaryotes, the tRNA genes exist as multiple copies and are transcribed by RNA polymerase III (RNA Pol III). As in prokaryotes, several tRNAs may be transcribed together to yield a single pre-tRNA molecule that is then processed to release the mature tRNAs. The promoters of eukaryotic tRNA genes are unusual in that the transcriptional control elements are located downstream (i.e. on the 3 side) of the transcriptional start site (at position +1). In fact they lie within the gene itself. Two such elements have been identified, called the A box and B box (Fig. 3). Transcription of the tRNA genes by RNA Pol III requires transcription factor IIIC (TFIIIC) as well as TFIIIB. THIIC binds to the A and B boxes whilst TFIIIB binds upstream of the A box. TFIIIB contains three subunits, one of which is TBP (TATA binding protein), the polypeptide required by all three eukaryotic RNA polymerases. 

Hepatic protein synthesis proceeds via the subcellular stages of gene transcription (in the nucleus) and gene translation (in the cytoplasm). The DNA is transcribed into various types of RNA by the action of the different DNA-dependent RNA polymerases I (A), II (B) and III (C). RNA polymerase I is responsible for the transcription of ribosomal RNA, RNA polymerase II mediates the transcription of messenger RNA, and RNA polymerase III forms transcriptal RNA. These three different RNA types move out of the nucleus into the cytoplasm. Here the ribosomes acquire the genetic information needed for protein synthesis via mRNA, and tRNA transports the activated amino acids to the ribosomes, which are themselves activated (and if necessary replicated) by rRNA. (s. figs. 2.9, 2.17 3.5)... 

Transcription is carried out by RNA polymerases, of which there are three types in the eukaryote. RNA pol I catalyzes the synthesis of rRNAs, RNA pol II is responsible for the synthesis of mRNA, and RNA pol III synthesizes tRNA. All three polymerases are large enzymes containing 12 or more subunits. 

The three eukaryotic RNA polymerases are distinguishable from one another by their differential sensitivity to the drag a-amanitin (the toxic principle of the mnshroom Amanita phalloides) which does not affect bacterial RNA polymerases. RNA polymerase II is very sensitive to a-amanitin, while RNA polymerase I is completely resistant. RNA polymerase III is moderately sensitive to this inhibitor. Mitochondria have yet another type of RNA polymerase, which is imaffected by a-amanitin bnt is sensitive to drags that inhibit bacterial RNA polymerase. A munber of antibiotics also act throngh their inhibition of transcription e.g., actinomycin D exerts its effect by binding to DNA templates, and it also blocks DNA replication. 

TBP is a key transcriptional factor required for transcriptional initiation by the three major RNA polymerases (RNAP I, II, and III) and is involved in gene expression of most eukaryotic genes. Expanding the polyQ stretch of TBP from 31Q into the pathogenic range of 71 Q reduced in vitro binding of TBP to the TATA box DNA [181]. In a SCA-17 mouse model, N-terminal TBP fragments are present, which harbor the expanded polyQ tract but lack an intact C-terminal... 

Another of the three subunits composing TFIIIB is TBP, which we can now see Is a component of a general transcription factor for all three eukaryotic nuclear RNA polymerases. The finding that TBP participates In transcription Initiation by Pol I and Pol III was surprising, since the promoters recognized by these enzymes often do not contain TATA boxes. Nonetheless, recent studies Indicate that the TBP subunit of TFIIIB Interacts with DNA similarly to the way It Interacts with TATA boxes. 

---