RNA Polymerase III Promoters

Amar, L., Desclaux, M., Faucon-Biguet, N., Mallet, J., and Vogel, R. (2006) Control of small inhibitory RNA levels and RNA interference by doxycycline induced activation of a minimal RNA polymerase III promoter. Nucl. Acids Res. 34, e37. 

RNA polymerase III promoters differ significantly from RNA polymerase II promoters in that they are located downstream from the transcription start site and within the transcribed segment of the DNA. For example, in the 5S RNA gene of the South African toad Xenopus laevis) the promoter is between 45 and 95 nucleotides downstream from the start point. Thus, the binding sites on RNA polymerase III are reversed with respect to the transcription direction, as compared with RNA polymerase II. That is, RNA polymerase II reaches forward to find the start point, and RNA polymerase III reaches backward. In fact, RNA polymerases can slide in either direction along a DNA template however, they can only synthesize RNA molecules in a 5 3 direction. 

Figure 29.17 Common eukaryotic promoter elements. Each eukaryotic RNA polymerase recognizes a set of promoier elements—sequences in DNA that promote transcription 1 he KNA polymerase I promoter consists of a ribosomal initator (rlnr) and an upstream promoter element (UPE). Tfie RNA polymerase II promotei likewise includes an initator element (Inr) and may also include either a TATA box or a downstream promoter dement (DPE), Separate from the promoter region, enhancer dements bind specific transcription factors. RNA polymerase III promoters consist of conserved sequences that lie within the transcribed genes.

TFIIIC subunits TFIIIC90 and TFIIICllO. The known function of TFIIIC is to initiate transcription complex formation by binding to promoter DNA and recruiting TBP-containing TFIIIB and RNA polymerase III, that directs synthesis of tRNA precursors. TFIIIC have an intrinsic HAT activity. 

What is unique about the promoter for RNA polymerase III Diagram how this promoter works. 

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. 

RNA polymerase III transcribes 5S rRNA and tRNA genes. The promoter of these transcripts can actually be located inside the gene itself, in contrast to all the other promoters discussed earlier. See Figure 12-10. 

The promoters for RNA polymerase III have been clearly proven to be internal on the template. In one instance the 5 S gene promoter lies between 45 and 83, whereas in tRNA genes it is split into two separate locations, one between 8 and 30 and a second between 51 and 72. Termination in 5 S genes is caused by a sequence of four A residues situated between two GC-rich regions. tRNA precursors are converted into mature tRNAs by a series of alterations (see later). 

Fig. 1. rRNA gene organization in the three domains. Abbreviations SSU, small subunit or 16S-like rRNA LSU, large subunit or 23S-like rRNA P, promoters Pi, promoter for RNA polymerase I P3, promoter for RNA polymerase III. In T. celer, the unlinked 5S RNA gene is part of an operon that includes an aspartic acid tRNA [26] while in M vannielii, the unlinked 5S RNA gene is part of an operon that includes 7 tRNA genes [27]. In Sulfolobus B12, Z). mobilis and T. tenax, the unlinked 5S rRNA is not part of an operon [28-31], Data from bacteria [23], archaea [24], eucarya[25], T. celer [26], M. vannielii [24, 27], Sulfolobus B12[28], D. mobilis[29], T. tenax [30], T. thermophilus [3 ], and T. acidophilum [32]. 

The design of shRNAs most commonly features a 19-21 bp duplex that is joined by a 4-9 nt loop sequence. The transcript is typically expressed by an RNA polymerase III (pol III) promoter, usually U6 or HI. Pol III promoters are ideal for this application for several reasons ... 

RNA polymerase III. Type I promoters, found in the 5S rRNA gene, con tain two short conserved sequences known as the. A block and the C Type II promoters, found in tRNA genes, consist of two 11-bp sequences, the A block and the B block, situated about 1 S bp from cither end ol the... 

Donze, D., and Kamakaka, R. T. (2001). RNA polymerase III and RNA polymerase II promoter complexes are heterochromatin barriers in Saccharomyces cerevisiae. EMBO J. 20, 520-531. 

Kassavetis, G. A., Bardeleben, C., Kumar, A., Ramirez, E., and Geiduschek, E. P. (1997). Domains of the Brf component of RNA polymerase III transcription factor IIIB (TFIIIB) functions in assembly of TFIIIB-DNA complexes and recruitment of RNA polymerase to the promoter. Mol. Cell. Biol. 17, 5299-5306. 

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