RNA polymerases (RNAPs) require basal transcription factors to assist them during transcription initiation. One of these factors, TFIIB, combines promoter recognition, recruitment of RNAP, promoter melting, start site selection and various post-initiation functions. The ability of 381 site-directed mutants in the TFIIB 'linker domain' to stimulate abortive transcription was systematically quantitated using promoter-independent dinucleotide extension assays. The results revealed two distinct clusters (mjTFIIB E78-R80 and mjTFIIB R90-G94, respectively) that were particularly sensitive to substitutions. In contrast, a short sequence (mjTFIIB A81-K89) between these two clusters tolerated radical single am... More
RNA polymerases (RNAPs) require basal transcription factors to assist them during transcription initiation. One of these factors, TFIIB, combines promoter recognition, recruitment of RNAP, promoter melting, start site selection and various post-initiation functions. The ability of 381 site-directed mutants in the TFIIB 'linker domain' to stimulate abortive transcription was systematically quantitated using promoter-independent dinucleotide extension assays. The results revealed two distinct clusters (mjTFIIB E78-R80 and mjTFIIB R90-G94, respectively) that were particularly sensitive to substitutions. In contrast, a short sequence (mjTFIIB A81-K89) between these two clusters tolerated radical single amino acid substitutions; short deletions in that region even caused a marked increase in the ability of TFIIB to stimulate abortive transcription ('superstimulation'). The superstimulating activity did, however, not correlate with increased recruitment of the TFIIB/RNAP complex because substitutions in a particular residue (mjTFIIB K87) increased recruitment by more than 5-fold without affecting the rate of abortive transcript stimulation. Our work demonstrates that highly localized changes within the TFIIB linker have profound, yet surprisingly disconnected, effects on RNAP recruitment, TFIIB/RNAP complex stability and the rate of transcription initiation. The identification of superstimulating TFIIB variants reveals the existence of a previously unknown rate-limiting step acting on the earliest stages of gene expression.
