The TFIIF Tfg1-E346A substitution causes upstream shifts in start site utilization on a variety of promoters in vivo. (A) Primer extension analysis of in vivo RNA. Total RNA isolated from yeast strains containing the indicated TFG1 and RPB9 alleles was analyzed by primer extension utilizing oligonucleotide primers specific for the indicated RNA transcripts. The positions of the major wild-type (WT) transcription start sites are indicated by the filled circles, while the open circles indicate the positions of upstream start sites exhibiting enhanced utilization in the mutant strains. (B) Shown are the promoter sequences for the above-described genes with the TATA elements underlined and the positions of the normal and upstream start sites indicated as in panel A.

The Tfg1-E346A mutant exhibits enhanced ability to utilize a suboptimal start site in vivo. (A) Primer extension analysis of in vivo transcripts produced from wild-type and variant ADH1 promoter constructs. Total RNA isolated from the wild-type (WT) or tfg1-E346A mutant strain harboring the indicated ADH1 promoter-HIS3 fusion construct was analyzed by primer extension utilizing a HIS3-specific oligonucleotide primer. The numbers to the left of the panel indicate the position of the transcription start sites, where +1 is defined as the A in the translation-initiating ATG codon. Arrow, position of the ADH1 −38 major transcript. (B) Sequences of the wild-type and variant ADH1 promoter constructs. Shown are the relevant sequences in the vicinity of the major ADH1 start sites for the wild-type promoter construct (plasmid p316/AH) and for the variant construct (plasmid p316/AH −48/43 A→T) that contains the highlighted (black boxes) nucleotide substitutions. The positions of the major start sites in the wild-type TFIIF strain are indicated by the filled circles; the open circles indicate the positions of the start sites in the tfg1-E346A mutant strain.

Both wild-type and Tfg1-E346A mutant TFIIF dramatically stabilize a 5-nucleotide RNA in the RNAPII active center. (A) Coomassie blue-stained 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis gel of the recombinant TFIIF preparations used in this study. Tfg1ΔN corresponds to a degradation product of Tfg1 lacking the N-terminal 80 to 90 amino acids. In vivo analyses have shown that the N-terminal 100 amino acids of Tfg1 are dispensable for cell viability and normal start site selection (unpublished data). Two additional minor degradation products of Tfg1 are highlighted with asterisks. (B) Mobility shift assays for DNA-RNAPII-TFIIF complexes. Reaction mixtures contained ³²P-labeled bubble80 template, purified wild-type or Rpb1-R344A RNAPII, and purified wild-type or Tfg1-E346A mutant TFIIF, as indicated. The positions of the DNA-RNAPII and the DNA-RNAPII-TFIIF complexes are shown at the left of the panel. (C) Mobility shift assays for DNA-RNAPII-TFIIF-RNA complexes. Reaction mixtures contained unlabeled bubble80 template, ³²P-labeled RNA5 oligonucleotide (5′-AGAGG-3′), purified wild-type or Rpb1-R344A RNAPII, and purified wild-type or Tfg1-E346A mutant TFIIF, as indicated. The positions of the DNA-RNAPII-RNA and DNA-RNAPII-TFIIF-RNA complexes are shown at the left of the panel.

Model for the role of yeast TFIIF in modulating conformational changes in the RNAPII active center during initiation and early elongation. Two TFIIF-RNAPII conformations are proposed. In conformation I, the binding of TFIIF results in a narrowing of the central cleft and involves key contacts between the Tfg1 subunit and the Rpb2 lobe that are stabilized by the adjacent Rpb9 subunit. Conformation I is characterized by a disordered switch 2, inefficient early bond formation, and nonprocessive elongation due to poor stabilization of the 3′ end of the RNA-DNA hybrid. In conformation II, the Tfg1 subunit has altered interaction with the Rpb2 lobe, and the Tfg2 subunit is repositioned in the central cleft. Importantly, conformation II is characterized by an ordered switch 2 positioned in the active center due to clamp closure, resulting in efficient early bond formation and processive elongation due to enhanced stabilization of the 3′ end of the RNA-DNA hybrid. Mutations in TFIIF and Rpb2 or deletion of Rpb9 that cause upstream shifts are proposed to destabilize conformation I and favor conformation II (highlighted with an asterisk), resulting in better utilization of suboptimal start sites closer to the TATA element. Approximate positions of the TFIIF domains that interact around the major cleft are highlighted in blue; additional potential interactions of TFIIF with other regions of RNAPII are not indicated.

Yeast PICs can encounter and utilize downstream start sites by a mechanism that does not require contiguous intervening RNA synthesis and that is not compromised by the Tfg1-E346A substitution. (A) Diagram of the ADH1 promoter/G-less cassette fusion template. Shown is the relevant portion of the plasmid template pADH1/G− that contains the ADH1 promoter fused to a 140-base-pair fragment of the G-less cassette (highlighted in black). The positions of the major ADH1 start site (designated −38 where +1 is defined as the A in the translation-initiating ATG codon) and an internal G-less cassette start site (designated with an asterisk) are indicated by the arrows. Highlighted in gray is a naturally occurring 14-base-pair G-less region in the vicinity of the major ADH1 start site, and the positions of upstream guanines are underlined. (B, C, and D) In vitro transcription reactions reconstituted with purified transcription factors. (B) Yeast PICs containing wild-type TFIIF were assembled on template pADH1/G− (lanes 2 to 4) or a TATA-lacking variant (lane 1), as described in Materials and Methods. Reactions were initiated by the addition of GTP-lacking (G−) NTPs in the presence of [α-³²P]CTP, and the products were either treated (lanes 1 to 3) or not (lane 4) with RNase T1 prior to electrophoresis. (C and D) Yeast PICs containing either wild-type (WT) or Tfg1-E346A mutant TFIIF were assembled on the pADH1/G− template, and reactions were initiated by the addition of GTP-containing (G+) or lacking (G−) NTPs in the presence of [α-³²P]CTP (C) or unlabeled CTP (D). Reaction products from the radiolabeled reactions were treated with RNase T1 prior to electrophoresis; reaction products from the unlabeled reactions were analyzed by primer extension. The numbers to the left of each panel correspond to the positions of the 5′ ends of the indicated transcripts.

Yeast TFIIF stimulates early phosphodiester bond formation, and this activity is increased by the Tfg1-E346A substitution. (A) Structure of the bubble80 template. Highlighted within the bubble are the locations of the template bases that give rise to a 5′-GGA-3′ trinucleotide product in the presence of GTP and radiolabeled ATP. (B) TFIIF stimulation of early phosphodiester bond formation. Reaction mixtures contained the bubble80 template, purified RNAPII, [α-³²P]ATP, unlabeled GTP, and purified wild-type or Tfg1-E346A mutant TFIIF as indicated. The 5′-GGA-3′ trinucleotide product is indicated by the arrow. (C) Effect of TFIIF on formation of the third phosphodiester bond. Reactions were performed as described in panel B except that GTP was replaced with the indicated amount of the unlabeled RNA3 oligonucleotide 5′-AGG-3′. The 5′-AGGA-3′ tetranucleotide product is indicated by the arrow. (D) Quantitation of TFIIF effects on early phosphodiester bond formation. Relative band intensities for the 5′-AGG-3′ and 5′-AGGA-3′ products for the gels shown in panels B and C were quantified using Bio-Rad QuantityOne software (n = 3).

The Tfg1-E346A mutant enhances processivity to a greater extent than wild-type (WT) TFIIF. Runoff transcription assays were performed as described in Materials and Methods with the bubble80 template; ³²P-labeled RNA5 (5′-AGAGG-3′); and purified wild-type or Rpb1-R344A RNAPII, TFIIS, and wild-type or Tfg1-E346A mutant TFIIF as indicated. Transcript lengths are shown at the left of the panel.