title: The elongation factor Spt4/5 regulates RNA polymerase II transcription through the nucleosome.
abstract: RNA polymerase II (RNAPII) passes through the nucleosome in a coordinated manner, generating several intermediate nucleosomal states as it breaks and then reforms histone-DNA contacts ahead of and behind it, respectively. Several studies have defined transcription-induced nucleosome intermediates using only RNA Polymerase. However, RNAPII is decorated with elongation factors as it transcribes the genome. One such factor, Spt4/5, becomes an integral component of the elongation complex, making direct contact with the 'jaws' of RNAPII and nucleic acids in the transcription scaffold. We have characterized the effect of incorporating Spt4/5 into the elongation complex on transcription through the 601R nucleosome. Spt4/5 suppressed RNAPII pausing at the major H3/H4-induced arrest point, resulting in downstream re-positioning of RNAPII further into the nucleosome. Using a novel single molecule FRET system, we found that Spt4/5 affected the kinetics of DNA re-wrapping and stabilized a nucleosomal intermediate with partially unwrapped DNA behind RNAPII. Comparison of nucleosomes of different sequence polarities suggest that the strength of the DNA-histone interactions behind RNAPII specifies the Spt4/5 requirement. We propose that Spt4/5 may be important to coordinate the mechanical movement of RNAPII through the nucleosome with co-transcriptional chromatin modifications during transcription, which is affected by the strength of histone-DNA interactions.
authors.affiliation: Department of Biochemistry and Molecular Biology, Center for Eukaryotic Gene Regulation, Penn State University, University Park, PA 16802, USA.
authors.forename: John B
authors.initials: JB
authors.lastname: Crickard
authors.affiliation: Department of Chemistry, Penn State University, University Park, PA 16802, USA.
authors.forename: Jaehyoun
authors.initials: J
authors.lastname: Lee
authors.affiliation: Department of Chemistry, Penn State University, University Park, PA 16802, USA.
authors.forename: Tae-Hee
authors.initials: TH
authors.lastname: Lee
authors.affiliation: Department of Biochemistry and Molecular Biology, Center for Eukaryotic Gene Regulation, Penn State University, University Park, PA 16802, USA.
authors.forename: Joseph C
authors.initials: JC
authors.lastname: Reese
mesh_terms: D002868:Chromosomal Proteins, Non-Histone / Q000502:physiology*; D004271:DNA, Fungal / Q000502:physiology; D015966:Gene Expression Regulation, Fungal; D009687:Nuclear Proteins / Q000502:physiology*; D009707:Nucleosomes / Q000502:physiology; D011485:Protein Binding; D012319:RNA Polymerase II / Q000235:genetics* / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics; D029701:Saccharomyces cerevisiae Proteins / Q000502:physiology*; D014158:Transcription, Genetic; D035602:Transcriptional Elongation Factors / Q000502:physiology*
chemical_list: D002868:Chromosomal Proteins, Non-Histone; D004271:DNA, Fungal; D009687:Nuclear Proteins; D009707:Nucleosomes; C080991:SPT4 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D035602:Transcriptional Elongation Factors; C069940:SPT5 transcriptional elongation factor; D012319:RNA Polymerase II