RNA Pol III genes: 5s and tRNA transcription
▶RNA polymerase III: RNA polymerase III (RNA Pol III) is a complex of at least 16 different subunits. Like RNA Pol II, it is located in the nucleoplasm. RNA Pol III synthesizes the precursors of 5S rRNA, the tRNAs and other snRNAs and cytosolic RNAs.
▶tRNA genes: The initial transcripts produced from tRNA genes are precursor molecules which are processed into mature RNAs . The transcription control regions of tRNA genes lie after the transcription start site within the transcription unit. There are two highly conserved sequences within the DNA encoding the tRNA, called the A box (5’-TGGCNNAGTGG-3’) and the B box (5’-GGTTCGANNCC-3’). These sequences also encode important sequences in the tRNA itself, called the D-loop and the TψC loop. This means that highly conserved sequences within the tRNAs are also highly conserved promoter DNA sequences.
Two complex DNA-binding factors have been identified which are required for tRNA transcription initiation by RNA Pol III . TFIIIC binds to both the A and B boxes in the tRNA promoter. TFIIIB binds 50 bp upstream from the A box. TFIIIB consists of three subunits, one of which is TBP, the general initiation factor required by all three RNA polymerases. The second is called BRF (TFIIB-related factor, since it has homology to TFIIB, the RNA Pol II initiation factor). The third subunit is called B’’. TFIIIB has no sequence specificity and therefore its binding site appears to be determined by the position of TFIIIC binding to the DNA. TFIIIB allows RNA Pol III to bind and initiate transcription. Once TFIIIB has bound, TFIIIC can be removed without affecting transcription. TFIIIC is therefore an assembly factor for the positioning of the initiation factor TFIIIB.
▶5S rRNA genes: RNA Pol III transcribes the 5S rRNA component of the large ribosomal subunit. This is the only rRNA subunit to be transcribed separately. Like the other rRNA genes which are transcribed by RNA Pol I, the 5S rRNA genes are tandemly arranged in a gene cluster. In humans, there is a single cluster of around 2000 genes. The promoters of 5S rRNA genes contain an internal control region called the C box which is located 81–99 bp downstream from the transcription start site. A second sequence termed the A box around bases +50 to +65 is also important.
The C box of the 5S rRNA promoter acts as the binding site for a specific DNA-binding protein, TFIIIA . TFIIIA acts as an assembly factor which allows TFIIIC to interact with the 5S rRNA promoter. The A box may also stabilize TFIIIC binding. TFIIIC is then bound to the DNA at an equivalent position relative to the start site as in the tRNA promoter. Once TFIIIC has bound, TFIIIB can interact with the complex and recruit RNA Pol III to initiate transcription.
▶Alternative RNA Pol III promoters: Many RNA Pol III genes also rely on upstream sequences for the regulation of their transcription. Some promoters such as the U6 small nuclear RNA (U6 snRNA) and small RNA genes from the Epstein–Barr virus use only regulatory sequences upstream from their transcription start sites. The coding region of the U6 snRNA has a characteristic A box. However, this sequence is not required for transcription. The U6 snRNA upstream sequence contains sequences typical of RNA Pol II promoters, including a TATA box at bases –30 to –23. These promoters also share several other upstream transcription factor binding sequences with many U RNA genes which are transcribed by RNA Pol II. These observations suggest that common transcription factors can regulate both RNA Pol II and RNA Pol III genes.
▶RNA Pol III termination: Termination of transcription by RNA Pol III appears only to require polymerase recognition of a simple nucleotide sequence. This consists of clusters of dA residues whose termination efficiency is affected by surrounding sequence. Thus the sequence 5_-GCAAAAGC-3_ is an efficient termination signal in the Xenopus borealis somatic 5S rRNA gene.
▶RNA polymerase III: RNA polymerase III (RNA Pol III) has 16 or more subunits. The enzyme is located in the nucleoplasm and it synthesizes the precursors of 5S rRNA, the tRNAs and other small nuclear and cytosolic RNAs.
▶tRNA genes: Two transcription control regions, called the A box and the B box, lie downstream from the transcription start site. These sequences are therefore both conserved sequences in tRNAs but also conserved promoter sequences in the DNA. TFIIIC binds to the A and B boxes in the tRNA promoter; TFIIIB binds to the TFIIIC–DNA complex and interacts with DNA upstream from the TFIIIC-binding site. TFIIIB contains three subunits, TBP, BRF and B’’, and is responsible for RNA Pol III recruitment and hence transcription initiation.
▶5S rRNA genes: The genes for 5S rRNA are organized in a tandem cluster. The 5S rRNA promoter contains a conserved C box 81–99 bases downstream from the start site, and a conserved A box at around 50–65 bases downstream. TFIIIA binds strongly to the C box promoter sequence. TFIIIC then binds to the TFIIIA–DNA complex, interacting also with the A box sequence. This complex allows TFIIIB to bind, recruit the polymerase, and initiate transcription.
▶Alternative RNA Pol III promoters: A number of RNA Pol III promoters are regulated by upstream as well as downstream promoter sequences. Other promoters require only upstream sequences, including the TATA box and other sequences found in RNA Pol II promoters.
▶RNA Pol III termination: The RNA polymerase can terminate transcription without accessory factors. A cluster of A residues is often sufficient for termination.