title: Structural basis of transcription initiation by bacterial RNA polymerase holoenzyme.
abstract: The bacterial RNA polymerase (RNAP) holoenzyme containing σ factor initiates transcription at specific promoter sites by de novo RNA priming, the first step of RNA synthesis where RNAP accepts two initiating ribonucleoside triphosphates (iNTPs) and performs the first phosphodiester bond formation. We present the structure of de novo transcription initiation complex that reveals unique contacts of the iNTPs bound at the transcription start site with the template DNA and also with RNAP and demonstrate the importance of these contacts for transcription initiation. To get further insight into the mechanism of RNA priming, we determined the structure of initially transcribing complex of RNAP holoenzyme with 6-mer RNA, obtained by in crystallo transcription approach. The structure highlights RNAP-RNA contacts that stabilize the short RNA transcript in the active site and demonstrates that the RNA 5'-end displaces σ region 3.2 from its position near the active site, which likely plays a key role in σ ejection during the initiation-to-elongation transition. Given the structural conservation of the RNAP active site, the mechanism of de novo RNA priming appears to be conserved in all cellular RNAPs.
authors: From the Department of Biochemistry and Molecular Biology, The Center for RNA Molecular Biology, The Pennsylvania State University, University Park, Pennsylvania 16802.
authors: Ritwika S
authors: RS
authors: Basu
authors: From the Department of Biochemistry and Molecular Biology, The Center for RNA Molecular Biology, The Pennsylvania State University, University Park, Pennsylvania 16802.
authors: Brittany A
authors: BA
authors: Warner
authors: From the Department of Biochemistry and Molecular Biology, The Center for RNA Molecular Biology, The Pennsylvania State University, University Park, Pennsylvania 16802.
authors: Vadim
authors: V
authors: Molodtsov
authors: the Laboratory of Molecular Genetics of Microorganisms, Institute of Molecular Genetics, Russian Academy of Sciences, Moscow 123182, Russia.
authors: Danil
authors: D
authors: Pupov
authors: the Laboratory of Molecular Genetics of Microorganisms, Institute of Molecular Genetics, Russian Academy of Sciences, Moscow 123182, Russia.
authors: Daria
authors: D
authors: Esyunina
authors: Centro de Investigaciones Biológicas, Consejo Superior de Investigaciones Científicas, Ramiro de Maeztu 9, 28040 Madrid, Spain, and.
authors: Carlos
authors: C
authors: Fernández-Tornero
authors: the Laboratory of Molecular Genetics of Microorganisms, Institute of Molecular Genetics, Russian Academy of Sciences, Moscow 123182, Russia.
authors: Andrey
authors: A
authors: Kulbachinskiy
authors: From the Department of Biochemistry and Molecular Biology, The Center for RNA Molecular Biology, The Pennsylvania State University, University Park, Pennsylvania 16802, kum14@psu.edu.
authors: Katsuhiko S
authors: KS
authors: Murakami
mesh_terms: D001483:Base Sequence; D001665:Binding Sites; D003460:Crystallization; D018360:Crystallography, X-Ray; D004269:DNA, Bacterial / Q000235:genetics; D012321:DNA-Directed RNA Polymerases / Q000737:chemistry / Q000378:metabolism*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic; D012689:Sequence Homology, Nucleic Acid; D016963:Thermus thermophilus / Q000201:enzymology; D024363:Transcription Initiation Site
chemical_list: D004269:DNA, Bacterial; D012321:DNA-Directed RNA Polymerases
keywords: Promoter; RNA Polymerase; Transcription; Transcription Initiation Factor; X-ray Crystallography