Scanning mutagenesis reveals roles for helix N of the bacteriophage T7 RNApolymerase thumb subdomain in transcription complex stability, pausing, and termination

Deletions within the thumb subdomain (residues 335-408) of T7 RNA polymeras e decrease elongation complex stability and processivity, but the structure of a T7RNAP initial transcription complex containing a 3-nucleotide RNA re veals no interactions between the thumb and the RNA or DNA. Modeling of a l onger RNA in this structure, using a T7DNAP-primer-template structure as a guide, suggests that the phosphate ribose backbone of the RNA contacts a st retch of mostly positively charged side chains between residues 385 and 395 of helix N of the thumb. Scanning mutagenesis of this region reveals that alanine substitutions of Arg(391), Ser(393), and Arg(394) destabilize elong ation complexes and that substitutions at 393 and 394 increase termination of transcripts 5 or more bases in length. The alpha -carbons of all 3 of th ese residues lie on the side of helix N, which faces into the template-bind ing cleft of the RNA polymerase, and modeling suggests that they can contac t the RNA 4-5 bases away from the 3 ' -end. Alanine substitutions of other residues within 385-395 do not have marked effects on transcription complex stability, but alanine substitutions of Asp(388) and Tyr(385) reduce pausi ng and termination at the T7 concatemer junction , Both of these side chain s lie on the outer side of helix N, pointing away from the template binding cleft. The thumb subdomain of T7RNAP therefore has roles both in transcrip tion complex stabilization and in pausing and termination at the T7 concate mer junction.