Abstract
In Saccharomyces cerevisiae, RNA polymerase II (Pol II) selects transcription start sites (TSSs) by a unidirectional scanning process. During scanning, a preinitiation complex (PIC) assembled at an upstream core promoter initiates at select positions within a window ~40-120 bp downstream. Several lines of evidence indicate that Ssl2, the yeast homolog of XPB and an essential and conserved subunit of the general transcription factor (GTF) TFIIH, drives scanning through its DNA-dependent ATPase activity, therefore potentially controlling both scanning rate and scanning extent (processivity). To address questions of how Ssl2 functions in promoter scanning and interacts with other initiation activities, we leveraged distinct initiation-sensitive reporters to identify novel ssl2 alleles. These ssl2 alleles, many of which alter residues conserved from yeast to human, confer either upstream or downstream TSS shifts at the model promoter ADH1 and genome-wide. Specifically, tested ssl2 alleles alter TSS selection by increasing or narrowing the distribution of TSSs used at individual promoters. Genetic interactions of ssl2 alleles with other initiation factors are consistent with ssl2 allele classes functioning through increasing or decreasing scanning processivity but not necessarily scanning rate. These alleles underpin a residue interaction network that likely modulates Ssl2 activity and TFIIH function in promoter scanning. We propose that the outcome of promoter scanning is determined by two functional networks, the first being Pol II activity and factors that modulate it to determine initiation efficiency within a scanning window, and the second being Ssl2/TFIIH and factors that modulate scanning processivity to determine the width of the scanning widow.
Highlights
Transcription of eukaryotic protein-coding genes is carried out by RNA polymerase II (Pol II) in three sequential steps: initiation, elongation and termination[1]
Existing SSL2 alleles show transcription-dependent growth phenotypes and distinct transcription start sites (TSS) usage patterns To understand how TSSs are identified by promoter scanning and the potential roles for TFIIH, we first examined previously identified ssl[2] mutants[19, 42,43,44] for transcription-related phenotypes that we have demonstrated are predictive of specific initiation defects (Figure 1A)
We have previously shown that tested mutants that shift TSSs upstream due to altered promoter scanning result in an inability to express a functional IMD2 transcript, causing sensitivity to the IMPDH inhibitor mycophenolic acid (MPA)[13, 39, 41, 47] (Figure 1A, IMD2)
Summary
Transcription of eukaryotic protein-coding genes is carried out by RNA polymerase II (Pol II) in three sequential steps: initiation, elongation and termination[1]. Due to inability of magnetic-tweezers to detect DNA compaction in the particular setup used, how the Pol II machinery reaches downstream TSSs, whether through generation of a large bubble or translocation of a small bubble was not clear Both studies agree that an ATP-dependent PIC activity for promoter opening is likely Ssl[2] within TFIIH, which has been demonstrated as a DNA translocase within purified TFIIH in vitro. We found that hyperactive Pol II catalytic mutants shifted TSSs distributions upstream at promoters genome-wide, consistent with a higher probability of initiation at every TSS, and initiation happening on average earlier in the scanning process. How alterations to Ssl2/TFIIH translocase activity control TSS distributions has not been extensively investigated
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