RESUMEN
The trigger loop (TL) forms a conserved element in the RNA polymerase active centre that functions in the elongation phase of transcription. Here, we show that the TL also functions in transcription initiation and termination. Using recombinant variants of RNA polymerase from Pyrococcus furiosus and a reconstituted transcription system, we demonstrate that the TL is essential for initial RNA synthesis until a complete DNA-RNA hybrid is formed. The archaeal TL is further important for transcription fidelity during nucleotide incorporation, but not for RNA cleavage during proofreading. A conserved glutamine residue in the TL binds the 2'-OH group of the nucleoside triphosphate (NTP) to discriminate NTPs from dNTPs. The TL also prevents aberrant transcription termination at non-terminator sites.
Asunto(s)
Proteínas Arqueales/química , ARN Polimerasas Dirigidas por ADN/química , Elongación de la Transcripción Genética , Iniciación de la Transcripción Genética , Terminación de la Transcripción Genética , Secuencia de Aminoácidos , Proteínas Arqueales/genética , Proteínas Arqueales/metabolismo , Secuencia Conservada , ARN Polimerasas Dirigidas por ADN/genética , ARN Polimerasas Dirigidas por ADN/metabolismo , Datos de Secuencia Molecular , Mutación , Pyrococcus furiosus/enzimología , ARN/biosíntesis , División del ARN , Homología de Secuencia de AminoácidoRESUMEN
The lower jaws of archaeal RNA polymerase and eukaryotic RNA polymerase II include orthologous subunits H and Rpb5, respectively. The tertiary structure of H is very similar to the structure of the C-terminal domain of Rpb5, and both subunits are proximal to downstream DNA in pre-initiation complexes. Analyses of reconstituted euryarchaeal polymerase lacking subunit H revealed that H is important for open complex formation and initial transcription. Eukaryotic Rpb5 rescues activity of the DeltaH enzyme indicating a strong conservation of function for this subunit from archaea to eukaryotes. Photochemical cross-linking in elongation complexes revealed a striking structural rearrangement of RNA polymerase, bringing subunit H near the transcribed DNA strand one helical turn downstream of the active center, in contrast to the positioning observed in preinitiation complexes. The rearrangement of subunits H and A'' suggest a major conformational change in the archaeal RNAP lower jaw upon formation of the elongation complex.
Asunto(s)
Proteínas Arqueales/química , ARN Polimerasas Dirigidas por ADN/química , Subunidades de Proteína/química , Transcripción Genética , Proteínas Arqueales/metabolismo , Secuencia de Bases , ADN/química , ADN/metabolismo , ARN Polimerasas Dirigidas por ADN/metabolismo , Modelos Moleculares , Datos de Secuencia Molecular , Regiones Promotoras Genéticas , Subunidades de Proteína/metabolismo , ARN Polimerasa II/química , ARN Polimerasa II/metabolismoRESUMEN
To initiate gene transcription, RNA polymerase II (Pol II) requires the transcription factor IIB (B). Here we present the crystal structure of the complete Pol II-B complex at 4.3 A resolution, and complementary functional data. The results indicate the mechanism of transcription initiation, including the transition to RNA elongation. Promoter DNA is positioned over the Pol II active centre cleft with the 'B-core' domain that binds the wall at the end of the cleft. DNA is then opened with the help of the 'B-linker' that binds the Pol II rudder and clamp coiled-coil at the edge of the cleft. The DNA template strand slips into the cleft and is scanned for the transcription start site with the help of the 'B-reader' that approaches the active site. Synthesis of the RNA chain and rewinding of upstream DNA displace the B-reader and B-linker, respectively, to trigger B release and elongation complex formation.