TATA-binding Protein and Transcription Factor IIB Induce Transcript Slipping during Early Transcription by RNA Polymerase II

Abstract

To better understand the mechanism of steps in early transcription by RNA polymerase II (pol II), we investigated the molecular determinants of transcript slipping within complexes assembled on promoters containing a pre-melted transcription bubble from ؊9 to ؉3. Transcript slippage occurs when an RNA transcript contains a repetitive sequence that allows the transcript to slip back and pair with the template strand of the DNA at a new register before transcription continues. We established the contributions of individual transcription factors, DNA elements, and RNA length to slipping on a heteroduplex template using a highly purified human pol II transcription system. We found that transcripts slip at a very defined point in the transcription reaction, after pol II completes phosphodiester bond synthesis at register ؉5. This point is set by the position of the polymerase active site on the DNA template, as opposed to the length of the transcript, as well as by a repetitive CUCU sequence that must occur from ؉2 to ؉5. Interestingly, slipping at this juncture is induced by TATA-binding protein and transcription factor IIB and requires a TATA box but not a transcription factor IIB recognition sequence. We propose a model in which transcribing complexes, upon completing phosphodiester bond synthesis at register ؉5, enter one of two branches in which they either complete productive synthesis of the transcript or undergo multiple rounds of transcript slipping.Transcription of mRNA by pol 3 II is a complex process that involves the highly regulated interplay of promoter DNA, many protein factors, and the transcript RNA itself. The promoter DNA influences the level of transcription and the position at which RNA synthesis begins through sequences in the regulatory and core promoter elements (1, 2). In addition, the conformation of promoter DNA can regulate the level of transcription, both in the presence and absence of histones (3-7). The general transcription factors, including TFIIA, TFIIB, TFIID, TFIIE, TFIIF, and TFIIH, are required for pol II to transcribe in a specific fashion in most, if not all, genes (8). Regulated genespecific transcription in cells requires a plethora of additional factors, including activators, repressors, co-regulators, and chromatin-modifying factors (2,6,8,9). In addition to the promoter and protein factors, biochemical studies have shown that the RNA transcript is not simply the product of the reaction, but it also stabilizes elongation complexes through the RNA: DNA hybrid and influences transformations that occur during early transcription (3, 10, 11). Although the core factors required for pol II to synthesize a transcript in vitro are understood, structural and biochemical studies are still uncovering new information about the mechanism by which this process occurs and is regulated.Biochemical studies have shown that the transcription reaction is composed of a series of steps that minimally include the following: preinitiation complex formation, open complex formation, initiation, esc...

Minimal Promoter Systems Reveal the Importance of Conserved Residues in the B-finger of Human Transcription Factor IIB

“…In addition, the E51R:R66E double mutant did not produce the 11-nt transcript in the presence of CpA (lane 10). Like results reported recently with a similar system, slippage was greatly reduced by priming with the CpA dinucleotide (44). We conclude that the defect in transcriptional activity of mutations in residues Glu-51 and Arg-66 is independent of the ability to recognize the start site.…”

“…The major transcript from this promoter is a 10-nt G-less RNA. Longer and shorter minor transcripts are also present, indicating the "slippage" of the transcription complex that has been documented by others (43,44) and is dependent upon the presence of TBP and TFIIB (44).…”

Regulation of gene expression by reiterative transcription

“…Candidate macromolecules include RNA polymerase binding factors. Interestingly, recent studies revealed that transcript slippage during initiation by human RNA polymerase II can be induced by the TATA-binding protein and transcription factor TFIIB [9 •• ]. Other potential signals are factors that control transcription pausing, particularly during transcription elongation.…”

“…The mechanism apparently involves one or more rounds of a one-base upstream shift of the transcript so that the same nucleotide in the template specifies multiple residues in the transcript [7,8]. It has also been shown that reiterative transcription can occur within dinucleotide [9 •• ,10] and trinucleotide [11] repeat sequences in the template, apparently via two-base and three-base upstream shifts of the transcript, respectively. Reiterative transcription can occur during initiation, elongation, or termination and result in transcripts that are immediately released from the transcription complex [12,13 •• ] or are extended by normal elongation after a switch to nonreiterative nucleotide addition [14,15 •• ].…”

New insights into the role of TFIIB in transcription initiation

“…16 Several studies suggest a role for TFIIB in promoter escape by pol II. 11,14,17,18 In early pol II transcription, short DNA-RNA hybrids are unstable and are transiently ejected from the enzyme (abortive transcription initiation). It has been proposed that the TFIIB B-finger/B-reader loop stabilizes the short RNA transcripts with pol II until the growing RNA reaches seven nucleotides and TFIIB is triggered to release from the initiation apparatus as pol II enters the productive elongation complex (promoter escape).…”