The linker domain of basal transcription factor TFIIB controls distinct recruitment and transcription stimulation functions

Abstract

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.

Molecular Mechanisms of Transcription Initiation—Structure, Function, and Evolution of TFE/TFIIE-Like Factors and Open Complex Formation

“…The TFB B-reader and B-helix motifs, which connect the TFB ZR and -core domains, make intricate interactions with the inside of the RNAP clamp proximal to the active site. Mutational analysis of these structural elements in Mja and Pyrococcus furiosus (Pfu) TFB revealed conserved elements residing in the TFB linker region that are important for PIC stabilisation and synthesis of the initial phosphodiester bonds [29][30][31]. Is there more to the archaeal promoter than TATA and BRE?…”

Promoter Independent Abortive Transcription Assays Unravel Functional Interactions Between TFIIB and RNA Polymerase

“…Several yeast TFIIB-RNAP II co-crystals exist showing glimpses of the interactions between the TFIIB linker and the RNAP II surface and suggesting that the TFIIB linker penetrates the active centre cleft (22)(23)(24). Biochemical analyses demonstrated that the linker domain of TFIIB actively contributes to the catalytic activity of RNAP which in its presence is substantially higher than in its absence (25)(26)(27). The resolution of these RNAP II-TFIIB co-crystals is, however, poor and insufficient to either reveal or fully explain all the activities that TFIIB has in a PIC.…”

“…by substituting every amino acid residue of a given sequence by all 19 other amino acids) we were able to investigate the influence that TFIIB has on the catalytic activity of RNAP (26,27). This approach was facilitated by switching to a model system that at several occasions has proven its high degree of accessibility.…”

“…Saturation mutagenesis has given us extensive insight into the protein dynamics accompanying the nucleotide addition cycle catalysed by mjRNAP that structural data have failed to uncover (29,30). Given the success in this system, we recently included the interface of the mjTFIIB linker and mjRNAP into our studies to further investigate the stimulation effect of the linker on mjRNAP activity (26).…”

High-throughput Purification of Affinity-tagged Recombinant Proteins

“…High-throughput strategies and the automation of manual protocols on robotic liquid-handling platforms have created opportunities to perform such complex molecular biological procedures with little human intervention and minimal error rates [1][2][3][4][5] .…”