title: Biochemical Analysis of Yeast Suppressor of Ty 4/5 (Spt4/5) Reveals the Importance of Nucleic Acid Interactions in the Prevention of RNA Polymerase II Arrest.
abstract: RNA polymerase II (RNAPII) undergoes structural changes during the transitions from initiation, elongation, and termination, which are aided by a collection of proteins called elongation factors. NusG/Spt5 is the only elongation factor conserved in all domains of life. Although much information exists about the interactions between NusG/Spt5 and RNA polymerase in prokaryotes, little is known about how the binding of eukaryotic Spt4/5 affects the biochemical activities of RNAPII. We characterized the activities of Spt4/5 and interrogated the structural features of Spt5 required for it to interact with elongation complexes, bind nucleic acids, and promote transcription elongation. The eukaryotic specific regions of Spt5 containing the Kyrpides, Ouzounis, Woese domains are involved in stabilizing the association with the RNAPII elongation complex, which also requires the presence of the nascent transcript. Interestingly, we identify a region within the conserved NusG N-terminal (NGN) domain of Spt5 that contacts the non-template strand of DNA both upstream of RNAPII and in the transcription bubble. Mutating charged residues in this region of Spt5 did not prevent Spt4/5 binding to elongation complexes, but abrogated the cross-linking of Spt5 to DNA and the anti-arrest properties of Spt4/5, thus suggesting that contact between Spt5 (NGN) and DNA is required for Spt4/5 to promote elongation. We propose that the mechanism of how Spt5/NGN promotes elongation is fundamentally conserved; however, the eukaryotic specific regions of the protein evolved so that it can serve as a platform for other elongation factors and maintain its association with RNAPII as it navigates genomes packaged into chromatin.
authors.affiliation: From the Department of Biochemistry and Molecular Biology, Center for Eukaryotic Gene Regulation, Penn State University, University Park, Pennsylvania 16802 and.
authors.forename: J Brooks
authors.initials: JB
authors.lastname: Crickard
authors.affiliation: the Department of Biochemistry, Medical College of Wisconsin, Milwaukee, Wisconsin 53226.
authors.forename: Jianhua
authors.initials: J
authors.lastname: Fu
authors.affiliation: From the Department of Biochemistry and Molecular Biology, Center for Eukaryotic Gene Regulation, Penn State University, University Park, Pennsylvania 16802 and Jcr8@psu.edu.
authors.forename: Joseph C
authors.initials: JC
authors.lastname: Reese
mesh_terms: D002868:Chromosomal Proteins, Non-Histone / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D009687:Nuclear Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D009696:Nucleic Acids / Q000378:metabolism*; D011485:Protein Binding; D011487:Protein Conformation; D012319:RNA Polymerase II / Q000037:antagonists & inhibitors* / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000254:growth & development / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D014158:Transcription, Genetic; D035602:Transcriptional Elongation Factors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
chemical_list: D002868:Chromosomal Proteins, Non-Histone; D009687:Nuclear Proteins; D009696:Nucleic Acids; C080991:SPT4 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D035602:Transcriptional Elongation Factors; C069940:SPT5 transcriptional elongation factor; D012319:RNA Polymerase II
keywords: DNA Protein Interactions; RNA polymerase II; Spt4/5; gene transcription; transcription elongation factor; transcription factor; transcription regulation