Amino Acid Substitutions in Yeast TFIIF Confer Upstream Shifts in Transcription Initiation and Altered Interaction with RNA Polymerase II

Ghazy, Mohamed A.; Brodie, Seth A.; Ammerman, Michelle L.; Ziegler, Lynn; Ponticelli, Alfred S.

doi:10.1128/mcb.24.24.10975-10985.2004

Cited by 56 publications

(89 citation statements)

References 44 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Mutations in TFIIB, TFIIF, Rpb1, 2, and 9 that alter the accuracy of transcription start site selection have all been genetically identified 3,4,6,7,11,33,38 . It has been difficult to explain how mutations in Rpb2 lobe/protrusion and Rpb9 (Figs.…”

Section: Discussionmentioning

confidence: 99%

“…Thus, the combined results reveal the close association of TFIIB, TFIIF, TFIIE, and XPB and suggest important protein-protein interactions between these factors that are involved in postrecruitment steps. These steps would involve initial melting of the promoter DNA strands, insertion of the single stranded template strand into the active site of Pol, and translocation of the template strand through the active site of the enzyme until the transcription start site is recognized.Mutations in TFIIB, TFIIF, Rpb1, 2, and 9 that alter the accuracy of transcription start site selection have all been genetically identified 3,4,6,7,11,33,38 . It has been difficult to explain how mutations in Rpb2 lobe/protrusion and Rpb9 (Figs.…”

mentioning

confidence: 99%

See 1 more Smart Citation

The positions of TFIIF and TFIIE in the RNA polymerase II transcription preinitiation complex

Chen

Warfield

Hahn

2007

Nat Struct Mol Biol

158

187

View full text Add to dashboard Cite

The non-natural photoreactive amino acid p-Benzoyl-L-Phenylalanine (Bpa) was incorporated into the RNA polymerase (Pol) II surface surrounding the central cleft formed by the Rpb1 and Rpb2 subunits. Photocrosslinking of Preinitiation Complexes (PICs) with these Pol II derivatives and hydroxyl radical cleavage assays revealed that the TFIIF dimerization domain interacts with the Rpb2 lobe and protrusion domains adjacent to Rpb9 while TFIIE crosslinks to the Rpb1 clamp domain on the opposite side of the Pol II central cleft. Mutations in the Rpb2 lobe and protrusion domains were found to alter both Pol II-TFIIF binding and the transcription start site, a phenotype associated with mutations in TFIIF, Rpb9, and TFIIB. In combination with previous biochemical and structural studies, these new findings illuminate the structural organization of the PIC and reveal a network of protein-protein interactions involved in transcription start site selection.The earliest step in transcription initiation is recruitment of Pol II and the general transcription factors to form the Preinitiation Complex (PIC) 1 . Upon addition of ATP, the PIC transitions to the Open Complex state, resulting in separation of the DNA strands surrounding the transcription start site and insertion of the template strand into the active center of Pol II. Next, Pol II must locate the transcription start site, which in S. cerevisiae, can be over 60 base pairs distant from the initial site of DNA strand separation 2 . Mutations in the general factor TFIIF and in the B-finger domain of the general factor TFIIB can alter the transcription start site, suggesting these two factors are involved in start site recognition 3-7 . Consistent with this role, crystallography and protein-protein crosslinking in the PIC have positioned both of these factors within the Pol II active site cleft 8-10 . Additionally, mutations in the Pol II subunit Rpb9 have been found to alter the transcription start site, but it remains unclear how this subunit, which is located distant from the Pol II active site, participates in start site selection 11,12 .Initiation of RNA synthesis begins with DNA-NTP base pairing, phosphodiester bond formation, and translocation of the DNA-RNA hybrid within the active center of Pol II. After synthesis of 8−12 bases of RNA, Pol II escapes from the promoter into a stable elongation state 13-15 . The nucleation of transcription factors at the promoter and the structural transition into the open and elongation complexes involves a complex set of protein-protein and protein-DNA interactions. Elucidating these molecular interactions within the transcription machinery Phone: 206 667 5261 Fax 206 667 6497 shahn@fhcrc.org. AUTHOR CONTRIBUTIONS L.W. modified the non-natural amino acid incorporation system and performed the experiment in Figure 1. H-T.C. performed and designed the remaining experiments. S.H. supervised the study. H-T.C. and S.H. wrote the manuscript.Publisher's Disclaimer: This PDF receipt will only be used as the basis for generating ...

show abstract

Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

The positions of TFIIF and TFIIE in the RNA polymerase II transcription preinitiation complex

Chen

Warfield

Hahn

2007

Nat Struct Mol Biol

158

187

View full text Add to dashboard Cite

show abstract

“…In contrast to the downstream shifts conferred by TFIIB mutations, we recently reported that mutations in either the Tfg1 or Tfg2 subunits of S. cerevisiae TFIIF can cause upstream shifts in the position of mRNA 5Ј-ends both in vivo and in vitro (12). Upstream shifts can also result from mutation or deletion of the small nonessential Rpb9 subunit of S. cerevisiae RNAPII (13)(14)(15).…”

mentioning

confidence: 87%

“…QuikChange site-directed mutagenesis (Stratagene) was employed to introduce amino acid substitutions into RPB1 contained on the TRP1-containing vector pRS314. The ability of the mutant plasmids to support viability was determined using the plasmid-shuffle complementation assay, and viable mutants were tested for conditional growth phenotypes as described previously (12). Complete lineages of all plasmids and strains used are available upon request.…”

Section: Methodsmentioning

confidence: 99%

“…Upstream shifts can also result from mutation or deletion of the small nonessential Rpb9 subunit of S. cerevisiae RNAPII (13)(14)(15). Although the precise mechanism by which these TFIIF or Rpb9 mutations cause upstream shifts remains unknown, biochemical analyses have shown that these mutations alter TFIIF-RNAPII interaction and that the degree of impairment in this interaction conferred by a given mutation directly correlates with the severity in the degree of upstream shifts (12).…”

mentioning

confidence: 99%

See 1 more Smart Citation

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

Majovski

Khaperskyy

Ghazy

et al. 2005

Journal of Biological Chemistry

Self Cite

View full text Add to dashboard Cite

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...

show abstract

Structural and binding studies of the C‐terminal domains of yeast TFIIF subunits Tfg1 and Tfg2

et al. 2011

View full text Add to dashboard Cite

The general transcription factor TFIIF plays essential roles at several steps during eukaryotic transcription. While several studies have offered insights into the structure/function relationship in human TFIIF, much less is known about the yeast system. Here, we describe the first NMR structural and binding studies of the C-terminal domains (CTDs) of Tfg1 and Tfg2 subunits of Saccharomyces cerevisiae TFIIF. We used the program CS-ROSETTA to determine the three-dimensional folds of these domains in solution, and performed binding studies with DNA and protein targets. CS-ROSETTA models indicate that the Tfg1 and Tfg2 C-terminal domains have winged-helix architectures, similar to the human homologs. We showed that both Tfg1 and Tfg2 CTDs interact with double-stranded DNA oligonucleotides, and mapped the DNA binding interfaces using solution NMR. Tfg1-CTD, but not Tfg2-CTD, also binds to yeast FCP1, an RNA polymerase II-specific phosphatase, and we delineated the interaction surface with the CTD of FCP1. Our results provide insights into the structural basis of yeast TFIIF function and the differential roles of Tfg1 and Tfg2 subunits during transcription.

show abstract

Amino Acid Substitutions in Yeast TFIIF Confer Upstream Shifts in Transcription Initiation and Altered Interaction with RNA Polymerase II

Cited by 56 publications

References 44 publications

The positions of TFIIF and TFIIE in the RNA polymerase II transcription preinitiation complex

The positions of TFIIF and TFIIE in the RNA polymerase II transcription preinitiation complex

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

Structural and binding studies of the C‐terminal domains of yeast TFIIF subunits Tfg1 and Tfg2

Contact Info

Product

Resources

About