High-resolution mutagenesis of the linker domain of archaeal basal transcription factor TFIIB

Abstract

TFIIB is a component of the minimal eukaryotic as well as the archaeal transcriptional machinery that is essential for promoter-directed transcription. The flexible linker domain of the protein engages in an intimate association with the RNA polymerase surface. Early structural data suggested that the linker forms a finger-like structure (the ‘B-finger’) within the RNA exit channel projecting into the active centre. Biochemical data indicated a contribution of the archaeal ‘B-finger’ domain to the catalytic mechanism by stimulating abortive transcription. The path of the linker within the RNA polymerase catalytic centre has recently been re-assessed and residues of the original Bfinger were re-assigned to structural elements named ‘B-reader helix’ and ‘B-reader loop’. Novel high-throughput tools, in combination with a comprehensive mutagenesis screen of residues E78 to A95, facilitated the biochemical evaluation of structural-functional relationships of the M. jannaschii TFIIB linker – RNAP interface at a single residue resolution. The performance of such point mutants during abortive initiation and RNA polymerase recruitment was interpreted in light of structural information. The tip region of the ‘B-finger’ that was predicted to be closest to the active site was insensitive to mutations in abortive initiation assays, thus disproving the original model. Individual residues, forming part of the B-reader helix and the C-terminal half of the Breader loop, were found to engage in abortive transcription. Three-residue deletions within the N-terminal half of the B-reader loop resulted in super-stimulation of abortive transcription. Individual point mutations within the B-reader loop led to enhanced recruitment of RNA polymerase. A functional role of the loop in stabilizing TFIIBRNA polymerase-DNA complexes in both the absence and presence of TBP seems feasible. The combined data provide a detailed view of biochemical functions of individual residues of the TFIIB linker favouring the ‘B-reader’ model over the ‘B-finger’ model