Region 3.2 of the σ Subunit Contributes to the Binding of the 3′-Initiating Nucleotide in the RNA Polymerase Active Center and Facilitates Promoter Clearance during Initiation

Abstract

Region 3.2 of the RNA polymerase subunit forms a loop that protrudes toward RNA polymerase active center and partially blocks RNA exit channel. To provide some insights into the functional role of this region, we studied a deletion variant of the Escherichia coli 70 subunit that lacked amino acids 513-519 corresponding to the tip of the loop. The deletion had multiple effects on transcription initiation including: (i) a significant decrease in the amount of short abortive RNAs synthesized during initiation, (ii) defects in promoter escape, (iii) loss of the contacts between the subunit and the nascent RNA during initiation and, finally, (iv) dramatic increase in the K m value for the 3-initiating nucleotide. At the same time, the mutation did not impair promoter opening and the binding of the 5-initiating purine nucleotide. In summary, our data demonstrate an important role of region 3.2 in the binding of initiating substrates in RNA polymerase active center and in the process of promoter clearance.The subunit of bacterial RNA polymerase (RNAP) 2 plays the key role in promoter-specific initiation of RNA synthesis. The subunit is involved in multiple processes during initiation including initial promoter recognition, DNA melting, abortive synthesis, and promoter clearance (1). Recent progress in structural studies of RNAP together with the bulk of biochemical data allowed the creation of an integral picture of transcription initiation and a proposal of functional roles for individual domains of the subunit. In RNAP holoenzyme, the subunit occupies the upstream part of the main RNAP channel. Two DNA binding domains of , involved in the recognition of the Ϫ10 and Ϫ35 promoter elements, are formed by conserved regions 2 and 4 and interact with a coiled-coil element of the ␤Ј subunit and the flap domain of the ␤ subunit, respectively (2-5). The promoter recognition domains of are connected by a flexible linker formed by conserved region 3.2 (amino acids 498 -526 in Escherichia coli numbering). A hairpin-like loop from region 3.2 protrudes toward the active center of RNAP and occupies a part of the RNA exit channel (Fig. 1). This led to several predictions about the functional role of this region. First, the region 3.2 loop was proposed to directly participate in the binding of the 5Ј-initiating nucleotide in the i-site of the RNAP active center (6, 7). In support of this, E. coli 70 was shown to cross-link to a 5Ј-initiating ATP analogue in a segment between amino acids 508 and 561 containing regions 3.2 and 4.1 (8). However, no experiments were done demonstrating the functional role for this region in substrate binding. Second, region 3.2 was proposed to play an important role in the process of promoter escape (3,6,7,9). As this region blocks the path for RNA exit (Fig. 1), the elongating RNA transcript must either displace it from the RNA exit channel thus promoting dissociation or dissociate itself from the complex and be released as an abortive product. Thus, direct competition between the elongating RNA transc...

Structure and function of the initially transcribing RNA polymerase II–TFIIB complex

“…The sigma factor is topologically related to TFIIB 1,2 and contains the loop region 3.2, which resembles the B-reader loop in location and negative charge [6][7][8] . Region 3.2 is required for formation of the first RNA phosphodiester bond, normal abortive transcription and sigma factor release [6][7][8] .…”

“…Once the RNA grows to 12-13 nucleotides, it clashes with TFIIB, triggering TFIIB displacement and elongation complex formation. Similar mechanisms may underlie all cellular transcription because all eukaryotic and archaeal RNA polymerases use TFIIB-like factors 5 , and the bacterial initiation factor sigma has TFIIB-like topology 1,2 and contains the loop region 3.2 that resembles the B-reader loop in location, charge and function [6][7][8] . TFIIB and its counterparts may thus account for the two fundamental properties that distinguish RNA from DNA polymerases: primer-independent chain initiation and product separation from the template.…”

Control of the Timing of Promoter Escape and RNA Catalysis by the Transcription Factor IIB Fingertip

“…As is the case with TFIIB, the sigma factor is also recycled during initiation (23). Region 3.2 of sigma has one property that has not been ascribed to TFIIB; namely, it abets efficient initiation and RNA bond formation by influencing the catalytic center of the RNA polymerase (21).…”

“…Several proteins that penetrate the active sites of RNA polymerases have regions that contain aspartates, and these strongly influence the catalytic properties of the enzyme. In the case of bacterial 70 , region 3.2 is required for efficient formation of an RNA bond during initiation (21). To assess whether the TFIIB fingertip plays a related catalytic role, we established an assay for initial RNA bond formation.…”

The TFIIB Tip Domain Couples Transcription Initiation to Events Involved in RNA Processing

“…There is some uncertainty about the structure of this region, and interconvertible structures may exist (5,7). Its configuration and placement within the preinitiation complex suggest that it may serve as an RNA placeholder and as the RNA grows during initiation the region is displaced, which contributes to the release of TFIIB, similar to the role of domains within bacterial sigma factor (32,33).…”