Abstract
The B block promoter element is the primary binding site for the RNA polymerase III transcription initiation factor TFIIIC. It is always located within the transcript coding region, except in the Saccharomyces cerevisiae U6 RNA gene (SNR6), in which the B block lies 120 base pairs downstream of the terminator. We have exploited the unique location of the SNR6 B block to examine the sequence specificity of its interaction with TFIIIC. The in vitro and in vivo effects of all possible single base pair substitutions in the 9-base pair core of the B block were determined. Five mutant alleles are recessive lethal when present at a low copy number; these alleles identify crucial contacts between TFIIIC and the B block promoter element. Transcript analysis reveals that lethal B block substitutions reduce U6 RNA synthesis at least 10-fold in vivo and 20-fold in vitro. One viable B block mutant strain has one-third the wild type amount of U6 RNA and exhibits reduced levels of the U4-U6 RNA complex required for spliceosome assembly. The locations of lethal single and double point mutations leads us to propose that two domains of TFIIIC contact overlapping sites on the B block element.
Highlights
The B block promoter element is the primary binding site for the RNA polymerase III transcription initiation factor TFIIIC
The most common class of genes transcribed by RNA polymerase III (RNAPIII),l which includes the tRNA genes and other cellular and viral small RNA genes, has a promoter that consists of two intragenic elements called the A and B blocks
The A and B block sequences apparently first functioned as RNA elements and were later recruited as promoter elements, since they are present in prokaryotic tRNA genes
Summary
The B block promoter element is the primary binding site for the RNA polymerase III transcription initiation factor TFIIIC. It is always located within the transcript coding region, except in the Saccharomyces cerevisiae U6 RNA gene (SNR6), in which the B block lies 120 base pairs downstream of the terminator. Unlike tRNA genes, SNR6 contains a consensus TATA box 30 base pairs upstream of the transcription start site. In vivo analysis ofthe promoter elements ofSNR6 revealed that the A and B blocks are required for transcription, while the TATA box and sequences upstream of it are not, complete substitution of the TATA box does alter start site selection in vivo [12]. The SNR6 transcription initiation complex is likely to be similar to that which assembles on a tRNA gene and unlike the vertebrate U6 RNA gene transcription complex
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