Unphosphorylated SR-Like Protein Npl3 Stimulates RNA Polymerase II Elongation

The production of a functional mRNA is regulated at every step of transcription. An area not well-understood is the transition of RNA polymerase II from elongation to termination. The S. cerevisiae SR-like protein Npl3 functions to negatively regulate transcription termination by antagonizing the binding of polyA/termination proteins to the mRNA. In this study, Npl3 is shown to interact with the CTD and have a direct stimulatory effect on the elongation activity of the polymerase. The interaction is inhibited by phosphorylation of Npl3. In addition, Casein Kinase 2 was found to be required for the phosphorylation of Npl3 and affect its ability to compete against Rna15 (Cleavage Factor I) for binding to polyA signals. Our results suggest that phosphorylation of Npl3 promotes its dissociation from the mRNA/RNAP II, and contributes to the association of the polyA/termination factor Rna15. This work defines a novel role for Npl3 in elongation and its regulation by phosphorylation

Yeast and Human RNA Polymerase II Elongation Complexes: Evidence for Functional Differences and Postinitiation Recruitment of Factors

Immobilized DNA templates, glycerol gradient centrifugation, and native gel analysis were utilized to isolate and compare functional RNA polymerase II (RNAPII) elongation complexes from Saccharomyces cerevisiae and human cell nuclear extracts. Yeast elongation complexes blocked by incorporation of 3′-O-methyl-GTP into the nascent transcript exhibited a sedimentation coefficient of 35S, were less tightly associated to the template than their human counterparts, and displayed no detectable 3′-5′ exonuclease activity on the associated transcript. In contrast, blocked human elongation complexes were more tightly bound to the template, and multiple forms were identified, with the largest exhibiting a sedimentation coefficient of 60S. Analysis of the associated transcripts revealed that a subset of the human elongation complexes exhibited strong 3′-5′ exonuclease activity. Although isolated human preinitiation complexes were competent for efficient transcription, their ability to generate 60S elongation complexes was strikingly impaired. These findings demonstrate functional and size differences between S. cerevisiae and human RNAPII elongation complexes and support the view that the formation of mature elongation complexes involves recruitment of nuclear factors after the initiation of transcription

Functions of Saccharomyces cerevisiae TFIIF during Transcription Start Site Utilization ▿

Previous studies have shown that substitutions in the Tfg1 or Tfg2 subunits of Saccharomyces cerevisiae transcription factor IIF (TFIIF) can cause upstream shifts in start site utilization, resulting in initiation patterns that more closely resemble those of higher eukaryotes. In this study, we report the results from multiple biochemical assays analyzing the activities of wild-type yeast TFIIF and the TFIIF Tfg1 mutant containing the E346A substitution (Tfg1-E346A). We demonstrate that TFIIF stimulates formation of the first two phosphodiester bonds and dramatically stabilizes a short RNA-DNA hybrid in the RNA polymerase II (RNAPII) active center and, importantly, that the Tfg1-E346A substitution coordinately enhances early bond formation and the processivity of early elongation in vitro. These results are discussed within a proposed model for the role of yeast TFIIF in modulating conformational changes in the RNAPII active center during initiation and early elongation

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

Transcription factor IIF (TFIIF) is required for transcription of protein-encoding genes by eukaryotic RNA polymerase II. In contrast to numerous studies establishing a role for higher eukaryotic TFIIF in multiple steps of the transcription cycle, relatively little has been reported regarding the functions of TFIIF in the yeast Saccharomyces cerevisiae. In this study, site-directed mutagenesis, plasmid shuffle complementation assays, and primer extension analyses were employed to probe the functional domains of the S. cerevisiae TFIIF subunits Tfg1 and Tfg2. Analyses of 35 Tfg1 alanine substitution mutants and 19 Tfg2 substitution mutants identified 5 mutants exhibiting altered properties in vivo. Primer extension analyses revealed that the conditional growth properties exhibited by the tfg1-E346A, tfg1-W350A, and tfg2-L59K mutants were associated with pronounced upstream shifts in transcription initiation in vivo. Analyses of double mutant strains demonstrated functional interactions between the Tfg1 mutations and mutations in Tfg2, TFIIB, and RNA polymerase II. Importantly, biochemical results demonstrated an altered interaction between mutant TFIIF protein and RNA polymerase II. These results provide direct evidence for the involvement of S. cerevisiae TFIIF in the mechanism of transcription start site utilization and support the view that a TFIIF-RNA polymerase II interaction is a determinant in this process

Transcription factors IIS and IIF enhance transcription efficiency by differentially modifying RNA polymerase pausing dynamics

Transcription factors IIS (TFIIS) and IIF (TFIIF) are known to stimulate transcription elongation. Here, we use a single-molecule transcription elongation assay to study the effects of both factors. We find that these transcription factors enhance overall transcription elongation by reducing the lifetime of transcriptional pauses and that TFIIF also decreases the probability of pause entry. Furthermore, we observe that both factors enhance the processivity of RNA polymerase II through the nucleosomal barrier. The effects of TFIIS and TFIIF are quantitatively described using the linear Brownian ratchet kinetic model for transcription elongation and the backtracking model for transcriptional pauses, modified to account for the effects of the transcription factors. Our findings help elucidate the molecular mechanisms by which transcription factors modulate gene expression

Genetic Interactions Between TFIIF and TFIIS

The eukaryotic transcript elongation factor TFIIS is encoded by a nonessential gene, PPR2, in Saccharomyces cerevisiae. Disruptions of PPR2 are lethal in conjunction with a disruption in the nonessential gene TAF14/TFG3. While investigating which of the Taf14p-containing complexes may be responsible for the synthetic lethality between ppr2Δ and taf14Δ, we discovered genetic interactions between PPR2 and both TFG1 and TFG2 encoding the two larger subunits of the TFIIF complex that also contains Taf14p. Mutant alleles of tfg1 or tfg2 that render cells cold sensitive have improved growth at low temperature in the absence of TFIIS. Remarkably, the amino-terminal 130 amino acids of TFIIS, which are dispensable for the known in vitro and in vivo activities of TFIIS, are required to complement the lethality in taf14Δ ppr2Δ cells. Analyses of deletion and chimeric gene constructs of PPR2 implicate contributions by different regions of this N-terminal domain. No strong common phenotypes were identified for the ppr2Δ and taf14Δ strains, implying that the proteins are not functionally redundant. Instead, the absence of Taf14p in the cell appears to create a dependence on an undefined function of TFIIS mediated by its N-terminal region. This region of TFIIS is also at least in part responsible for the deleterious effect of TFIIS on tfg1 or tfg2 cold-sensitive cells. Together, these results suggest a physiologically relevant functional connection between TFIIS and TFIIF