Biology subjects

Yuzenkova, Y.

Publications and source records attributed to Yuzenkova, Y..

7 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↗

bioRxiv2025

Canonical transcription termination mechanisms explain a minority of operons in cyanobacteria

Cyanobacteria are the most abundant phototrophs and hold potential as a carbon-negative platform for bioengineering applications. However, these efforts have been hampered by limited mechanistic understanding of their gene expression, including transcription termination. Unlike most bacteria, cyanobacteria lack the transcription termination factor Rho, raising the speculation that all transcription ends with intrinsic terminators. Here we show that most transcription units (TUs) in Synechococcus elongatus PCC 7942 are not terminated by known termination pathways. Although many TUs (52%) have unique, well-defined 3' ends, only a small fraction have features that resemble canonical intrinsic terminators (22%). The noncanonical 3' ends broadly lacked strong secondary structure, making it unclear how these ends are protected against 3'-5' exonucleolytic decay. Furthermore, many TUs (46%) have diverse positions of mRNA 3' ends, suggesting a potentially diffuse termination signal. Finally, we observed a moderate increase in RNA levels downstream of most defined 3' ends in the absence of the transcription-repair coupling factor Mfd. This finding indicates that Mfd plays a limited, but widespread, role in RNA end formation, potentially through termination of stalled RNAPs. Together, our work reveals unique end architectures of the cyanobacterial transcriptome and suggests that undescribed transcription termination mechanisms are active in the phylum. ImportanceOur understanding of bacterial transcription regulation is largely based on model organisms like Escherichia coli and Bacillus subtilis, yet many of these mechanisms appear absent or divergent in cyanobacteria. These differences limit our fundamental understanding of gene regulation and the applied potential of cyanobacteria in sustainable biomanufacturing. To address this gap, we characterized transcription termination in the model cyanobacterium Synechococcus elongatus PCC 7942. We resolve a longstanding question by showing that intrinsic termination alone cannot account for most termination events in this organism. Pervasive transcript ends lacking intrinsic terminator features and the absence of Rho suggest the existence of novel termination mechanism(s) and highlight a largely unexplored regulatory landscape. Simultaneously, our work expands the repertoire of functionally characterized cyanobacterial intrinsic terminators, offering a new toolkit to fine-tune gene expression using terminators of defined strengths. These findings pave the way for more predictable and powerful applications of cyanobacteria in green biotechnology.

microbiology↗

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↗

bioRxiv2023

5'-NADylation of RNA is well-tolerated by bacterial cell

Recently discovered ability of various RNA polymerases (RNAPs) of bacteria and eukaryotes to initiate RNA synthesis with cofactors such as NAD+/NADH (nicotinamide adenine dinucleotide). This phenomenon was quickly dubbed "non-canonical capping" as the extra 5 moieties of RNA resemble eukaryotic m7G cap superficially. NAD must outcompete abundant ATP, to serve as initiating nucleotide. It is still unclear which physiological conditions permit significant RNA NADylation and to what degree NADylation serves typical functions of a canonical cap in bacteria. Here we found that the extent of NADyation depends primarily on fidelity of transcription initiation -NAD concentration, specific contacts of RNAP active site with NAD/ATP alternative substrates, and genomic DNA supercoiling. It is much less affected by posttranscriptional processes such as de-capping, processing by main 5-dependent ribonucleases RNaseE and oligoribonuclease. 5-NAD inhibits neither translation of a leaderless 5-NAD-RNA nor a physiological base-pairing of small regulatory NADylated RNA with its antisense target RNA. Translation exposes 5-NAD for de-capping by NudC enzyme.

molecular biology↗

bioRxiv2022

A SI3-sigma; arch stabilizes cyanobacteria transcription initiation complex

Multi-subunit RNA polymerases (RNAPs) associate with initiation factors ({sigma} in bacteria) to start transcription. The {sigma} factors are responsible for recognizing and unwinding promoter DNA in all bacterial RNAPs. Here, we report two cryo-EM structures of cyanobacterial transcription initiation complexes at near-atomic resolutions. The structures show that cyanobacterial RNAP forms an SI3-{sigma} arch interaction between domain 2 of {sigma}A ({sigma}2) and the sequence insertion 3 (SI3) in the mobile catalytic domain Trigger Loop (TL). The SI3-{sigma} arch facilitates transcription initiation from promoters of different classes through sealing the main cleft and thereby stabilizing RNAP-promoter DNA open complex. Disruption of the SI3-{sigma} arch disturbs cyanobacteria growth and stress response. Our study reports the structure of cyanobacterial RNAP and unique mechanism for its transcription initiation. Our data suggest functional plasticity of SI3 and provide foundation for further research into cyanobacteria and chloroplasts transcription.

biochemistry↗

bioRxiv2019

Metabolic cofactors act as initiating substrates for primase and affect replication primer processing

Recently a new, non-canonical type of 5-RNA capping with cellular metabolic cofactors was discovered in bacteria and eukaryotes. This type of capping is performed by RNA polymerases, the main enzymes of transcription, which initiate RNA synthesis with cofactors. Here we show that primase, the enzyme of replication which primes synthesis of DNA by making short RNA primers, initiates synthesis of replication primers using the number of metabolic cofactors. Primase DnaG of E. coli starts synthesis of RNA with cofactors NAD+/NADH, FAD and DP-CoA in vitro. This activity does not affect primase specificity of initiation. ppGpp, the global starvation response regulator, strongly inhibits the non-canonical initiation by DnaG. Amino acid residues of a \"basic ridge\" define the binding determinant of cofactors to DnaG. Likewise, the human primase catalytic subunit P49 can use modified substrate m7GTP for synthesis initiation.\n\nFor correct genome duplication, the RNA primer needs to be removed and Okazaki fragments ligated. We show that the efficiency of primer processing by DNA polymerase I is strongly affected by cofactors on the 5-end of RNA. Overall our results suggest that cofactors at the 5 position of the primer influence regulation of initiation and Okazaki fragments processing.\n\nVisual abstract O_FIG_DISPLAY_L [Figure 1] M_FIG_DISPLAY A. Non-canonical capping of RNA by RNA polymerase. RNA polymerase uses cellular cofactor as initiating substrate for RNA synthesis, instead of NTP. Then RNA chain grows, while cofactor remains attached and serves as cap. B. Proposed mechanism of non-canonical initiation of RNA primer synthesis by DnaG primase during replication. DnaG primase initiates synthesis of the primer for DNA replication using cellular cofactor. Primer stays annealed with the DNA template. DNApolI encounters cofactor, which affects the removal of primer.\n\nC_FIG_DISPLAY

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↗