Biology subjects

Riaz-Bradley, A.

Publications and source records attributed to Riaz-Bradley, A..

3 recordsLinked to original sources

bioRxiv2025

Intrinsic RNA hairpin-mediated transcription termination at high temperature in Thermus aquaticus

Transcription termination establishes gene boundaries and limits regulatory interference. In bacteria, intrinsic termination, mediated by an RNA hairpin followed by a polyuridine tract (polyU), is a principal mechanism. Because RNA secondary structures are destabilized at high temperature, whether intrinsic termination functions effectively in thermophiles has been uncertain. We mapped RNA 3' termini genome-wide in the extreme thermophile Thermus aquaticus (Taq) grown at 70 {degrees}C using Term-Seq. Computational analysis identified hairpins upstream of most termini. These hairpins were substantially more thermally stable than those of the mesophile Escherichia coli. RNA 3' ends were also detected within coding regions, consistent with transcriptional pausing or premature termination at spurious hairpins in GC-rich transcripts. In vitro transcription showed that native Taq terminators direct efficient intrinsic termination by Taq RNA polymerase at 70 {degrees}C and require both hairpin and polyU. Termination remained efficient up to 80 {degrees}C. At this temperature, the terminator hairpin RNA is unfolded in solution, indicating that RNA polymerase promotes hairpin formation during transcription. Thus, intrinsic termination operates efficiently in thermophiles. At lower temperatures, expression of the Rho termination factor increased, suggesting that factor-dependent termination may contribute more prominently under cooler growth conditions in Thermus. Significance StatementIntrinsic transcription termination requires formation of an RNA hairpin. Hairpin structures are expected to be unstable at the high temperatures inhabited by thermophilic bacteria. Whether and how hairpin-dependent termination operates under these conditions has remained unknown. Using genome-wide transcripts ends mapping and biochemical approaches, we show that the RNA polymerase of Thermus aquaticus promotes folding of the nascent RNA into a terminator hairpin. This enables efficient termination even at extreme temperatures. Raising temperature favours entropy-driven dissociation of the transcription complex. At lower temperatures, termination appears to increasingly rely on a factor-dependent pathway. Together, these results identify RNA polymerase as an active participant in hairpin-dependent termination and show how temperature may shift the balance between alternative termination mechanisms.

molecular biology↗

bioRxiv2024

Structure and function of the Si3 insertion integrated into the trigger loop/helix of cyanobacterial RNA polymerase

Cyanobacteria and evolutionarily related chloroplasts of algae and plants possess unique RNA polymerases (RNAPs) with characteristics that distinguish from canonical bacterial RNAPs. The largest subunit of cyanobacterial RNAP (cyRNAP) is divided into two polypeptides, {beta}1 and {beta}2, and contains the largest known lineage-specific insertion domain, Si3, located in the middle of the trigger loop and spans approximately half of the {beta}2 subunit. In this study, we present the X-ray crystal structure of Si3 and the cryo-EM structures of the cyRNAP transcription elongation complex plus the NusG factor with and without incoming nucleoside triphosphate (iNTP) bound at the active site. Si3 has a well-ordered and elongated shape that exceeds the length of the main body of cyRNAP, fits into cavities of cyRNAP and shields the binding site of secondary channel-binding proteins such as Gre and DksA. A small transition from the trigger loop to the trigger helix upon iNTP binding at the active site results in a large swing motion of Si3; however, this transition does not affect the catalytic activity of cyRNAP due to its minimal contact with cyRNAP, NusG or DNA. This study provides a structural framework for understanding the evolutionary significance of these features unique to cyRNAP and chloroplast RNAP and may provide insights into the molecular mechanism of transcription in specific environment of photosynthetic organisms. Significance statementCellular RNA polymerase (RNAP) carries out RNA synthesis and proofreading reactions utilizing a mobile catalytic domain known as the trigger loop/helix. In cyanobacteria, this essential domain acquired a large Si3 insertion during the course of evolution. Despite its elongated shape and large swinging motion associated with the transition between the trigger loop and helix, Si3 is effectively accommodated within cyRNAP, with no impact on the fundamental functions of the trigger loop. Understanding the significance of Si3 in cyanobacteria and chloroplasts is expected to reveal unique transcription mechanism in photosynthetic organisms.

biochemistry↗

bioRxiv2019

RNA polymerase active centre compensates for the absence of transcription proofreading factors in Cyanobacteria

The vast majority of organisms possess transcription elongation factors, the functionally similar bacterial Gre and eukaryotic TFIIS/TFS. Their main cellular functions are to proofread errors of transcription and to restart elongation via stimulation of RNA hydrolysis by the active centre of RNA polymerase (RNAP). Very few taxons lack these factors, including the large evolutionarily ancient group of cyanobacteria and their descendants, the chloroplasts. How do they cope? What compensatory mechanisms they possess?\n\nWe found that cyanobacterial RNAP functionally substitutes for Gre/TFIIS - it does not stall on DNA, it efficiently catalyses the proofreading reaction of RNA hydrolysis, and the drop in transcription fidelity is only fractional, as confirmed by NGS. This alternative, presumably primordial, route to fidelity and processivity in the absence of Gre/TFIIS factors is based on the active site of RNAP stabilisation in a closed conformation. However, here lies a trade off - a severely reduced ability of this active site to recognise regulatory pausing signals. We suggest that perhaps the main advantage of Gre/TFIIS acquisition was to allow transcription regulation via pausing; with increase in fidelity as a bonus side effect.

biochemistry↗