Region 3.2 of the sigma subunit contributes to the binding of the 3'-initiating nucleotide in the RNA polymerase active center and facilitates promoter clearance during initiation.
Region 3.2 of the RNA polymerase sigma subunit forms a loop that protrudes toward RNA polymerase active center and partially blocks RNA exit channel. To provide some insights into the functional role of this region, we studied a deletion variant of the Escherichia coli sigma(70) subunit that lacked amino acids 513-519 corresponding to the tip of the loop. The deletion had multiple effects on transcription initiation including: (i) a significant decrease in the amount of short abortive RNAs synthesized during initiation, (ii) defects in promoter escape, (iii) loss of the contacts between the sigma subunit and the nascent RNA during initiation and, finally, (iv) dramatic increase in the K(m) value for the 3'-initiating nucleotide. At the same time, the mutation did not impair promoter opening and the binding of the 5'-initiating purine nucleotide. In summary, our data demonstrate an important role of sigma region 3.2 in the binding of initiating substrates in RNA polymerase active center and in the process of promoter clearance.
Kulbachinskiy
Andrey
A
Institute of Molecular Genetics, Russian Academy of Sciences, Moscow 123182, Russia. akulb@img.ras.ru
Mustaev
Arkady
A
D001665:Binding Sites / Q000235:genetics; D012321:DNA-Directed RNA Polymerases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism; D015964:Gene Expression Regulation, Bacterial; D008958:Models, Molecular; D011401:Promoter Regions, Genetic / Q000235:genetics*; D017384:Sequence Deletion; D012808:Sigma Factor / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D013329:Structure-Activity Relationship; D024363:Transcription Initiation Site; D014158:Transcription, Genetic
D029968:Escherichia coli Proteins; D012808:Sigma Factor; C081775:RNA polymerase sigma 70; D012321:DNA-Directed RNA Polymerases