A Functional Role for the Switch 2 Region of Yeast RNA Polymerase II in Transcription Start Site Utilization and Abortive Initiation

Abstract

RNA polymerase II (RNAPII) is responsible for the synthesis of mRNA from eukaryotic protein-encoding genes. In this study, sitedirected mutagenesis was employed to probe the function of residues within the Saccharomyces cerevisiae RNAPII active center in the mechanism of transcription start site utilization. We report here the identification of two mutations in the switch 2 region, rpb1-K332A and rpb1-R344A, which conferred conditional growth properties and downstream shifts in start site utilization. Analyses of double mutant strains demonstrated functional interactions between these switch 2 mutations and a mutation in the largest subunit of transcription factor IIF (TFIIF) that confers upstream shifts in start site usage. Importantly, biochemical analyses demonstrated that purified Rpb1-R344A mutant polymerase exhibited impaired ability to stabilize a short RNA-DNA hybrid in the active center, an increased frequency of abortive transcription in runoff assays, and both a downstream shift and increased abortive initiation in reconstituted transcription assays. These results provide evidence for a role of switch 2 during start site utilization and indicate that RNA-DNA hybrid stability at the 3-end of the transcript is a determinant in this process. We discuss these results within the context of a proposed model regarding the concerted roles of RNA-PII, TFIIB, and TFIIF during mRNA 5-end formation in S. cerevisiae.The synthesis of mRNA from protein-encoding (class II) genes in eukaryotic organisms is a regulated multistep process that involves the concerted action of RNA polymerase II (RNAPII) 2 and a host of auxiliary transcription factors (1, 2). In higher eukaryotes, transcription initiation typically occurs ϳ30 base pairs downstream of a TATA element. In contrast, mRNA 5Ј-ends in the yeast Saccharomyces cerevisiae frequently map to multiple sites within a window ranging from 40 to 120 base pairs or greater downstream of a TATA element (3,4). Although the precise mechanism by which RNAPII recognizes and productively utilizes a potential start site remains to be elucidated, previous studies have firmly established that RNAPII and the general transcription factors IIB (TFIIB) and IIF (TFIIF) play important roles in determining the position of mRNA 5Ј-ends in S. cerevisiae. Numerous genetic and biochemical studies of S. cerevisiae TFIIB conducted in our laboratory and by others have shown that amino acid substitutions in a highly conserved N-terminal region of TFIIB can alter the position of mRNA 5Ј-ends both in vivo and in vitro (5-10). Specifically, these TFIIB mutations confer a downstream shift in start site utilization, i.e. diminished utilization of sites located proximal to the TATA element with accompanying enhanced utilization of sites located further downstream. Interestingly, the structural determination of an S. cerevisiae TFIIB-RNAPII co-crystal has revealed that this N-terminal region of TFIIB adopts a finger-like structure that projects into the RNA exit channel of the polymerase and is positioned...

Schizosacharomyces pombe RNA polymerase II at 3.6-Å resolution

“…In addition, 2 loops in the Rpb2 lobe domain, ␤7-␤8 and ␤9-␤10, are shifted toward downstream DNA, which may affect pol II binding to TFIIF. Further along the cleft, the switch regions, important for binding the DNA-RNA hybrid, and start site selection (3,8,9), are structured in the absence of DNA in S. pombe, but not in S. cerevisiae. TFIIB, also important for start site selection, interacts with the pol II dock domain, which differs in structure between S. pombe and S. cerevisiae as well.…”

“…3B). The flipped-out region was in close proximity to the B finger in the pol II-TFIIB cocrystal structure (20) and contained the conserved residue Lys-338 Lys-332 shown to shift the transcription start site downstream upon mutation in S. cerevisiae (9). Mutations in switch 1 have been shown to shift the transcription start site downstream as well (8).…”

“…Conserved elements of the catalytic core include ''switch regions'' at the base of a swinging clamp, which interact with the DNA-RNA hybrid and downstream DNA during transcription (2,3) and may be involved in transcription start site determination (8,9). A so-called ''trigger loop'' interacts with the substrate nucleoside triphosphate to achieve the high fidelity of transcription (10)(11)(12)(13)(14).…”

Rethinking the role of TFIIF in transcript initiation by RNA polymerase II

“…15,19,20 This agrees with studies in yeast showing that TFIIF can influence start site selection through interactions with TFIIB. 12,[21][22][23][24] The presence of TFIIF within a yeast open complex significantly alters the location of TFIIB within that complex. 15 It has also been reported that TFIIF causes human RNP II to assume a particular orientation in complexes with Mediator.…”

Transcription factor TFIIF is not required for initiation by RNA polymerase II, but it is essential to stabilize transcription factor TFIIB in early elongation complexes

“…When TFIIB is in complex with pol II, a TFIIB domain referred to as the finger (9) or reader (10) enters pol II and approaches the catalytic center. Structural investigations of the yeast preinitiation complex (PIC) revealed a segment of TFIIF localized near the TFIIB domain that presumably directs transcription start site selection (7,11,12). It was suggested that this interaction with TFIIF is important in stabilizing TFIIB within the PIC (12).…”