Biology subjects

Kashlev, M.

Publications and source records attributed to Kashlev, M..

8 recordsLinked to original sources

bioRxiv2024

Nascent RNA profiling reveals regulation of gene transcription through productive reiterative initiation in bacteria

Productive reiterative initiation, an alternative transcription process accompanying RNA slippage relative to the template DNA and RNA polymerase, results in the incorporation of additional nucleotides into transcripts. However, a comprehensive understanding of the mechanisms underlying productive reiterative initiation and its functional implications has been hindered due to the complexity of heterogenous slippage transcripts. Here, we develop and employ 5 Terminal Native Elongating Transcript Sequencing (5TNET-seq) to identify and quantify productive reiterative initiation events in Escherichia coli. 5TNET-seq reveals that over 51% of promoters exhibit productive reiterative initiation. The conserved promoter - 10 region, an appropriate spacer between the -10 region and the TSS, and the transcription initiation region associated with weak RNA-DNA hybrid stability, particularly "AAA" and "TTT" trinucleotide tracts, contribute to high productive reiterative initiation. In addition, up to four nucleotides can be added in a single cycle of productive reiterative initiation. A smaller transcription bubble is observed during productive reiterative initiation, which may stabilize the transcription initiation complex to regulate gene transcription. Our results suggest that productive reiterative initiation emerges as an inherent transcription process regulating biological processes, including cell wall synthesis, independent of protein regulators.

microbiology↗

bioRxiv2024

Chromatin Buffers Torsional Stress During Transcription

Transcription through chromatin under torsion represents a fundamental problem in biology. Pol II must overcome nucleosome obstacles and, because of the DNA helical structure, must also rotate relative to the DNA, generating torsional stress. However, there is a limited understanding of how Pol II transcribes through nucleosomes while supercoiling DNA. In this work, we developed methods to visualize Pol II rotation of DNA during transcription and determine how torsion slows down the transcription rate. We found that Pol II stalls at {+/-} 9 pN{middle dot}nm torque, nearly sufficient to melt DNA. The stalling is due to extensive backtracking, and the presence of TFIIS increases the stall torque to + 13 pN{middle dot}nm, making Pol II a powerful rotary motor. This increased torsional capacity greatly enhances Pol IIs ability to transcribe through a nucleosome. Intriguingly, when Pol II encounters a nucleosome, nucleosome passage becomes more efficient on a chromatin substrate than on a single-nucleosome substrate, demonstrating that chromatin efficiently buffers torsional stress via its torsional mechanical properties. Furthermore, topoisomerase II relaxation of torsional stress significantly enhances transcription, allowing Pol II to elongate through multiple nucleosomes. Our results demonstrate that chromatin greatly reduces torsional stress on transcription, revealing a novel role of chromatin beyond the more conventional view of it being just a roadblock to transcription.

biophysics↗

bioRxiv2024

RNA Polymerase II is a Polar Roadblock to a Progressing DNA Fork

DNA replication and transcription occur simultaneously on the same DNA template, leading to inevitable conflicts between the replisome and RNA polymerase. These conflicts can stall the replication fork and threaten genome stability. Although numerous studies show that head-on conflicts are more detrimental and more prone to promoting R-loop formation than co-directional conflicts, the fundamental cause for the RNA polymerase roadblock polarity remains unclear, and the structure of these R-loops is speculative. In this work, we use a simple model system to address this complex question by examining the Pol II roadblock to a DNA fork advanced via mechanical unzipping to mimic the replisome progression. We found that the Pol II binds more stably to resist removal in the head-on configuration, even with minimal transcript size, demonstrating that the Pol II roadblock has an inherent polarity. However, an elongating Pol II with a long RNA transcript becomes an even more potent and persistent roadblock while retaining the polarity, and the formation of an RNA-DNA hybrid mediates this enhancement. Surprisingly, we discovered that when a Pol II collides with the DNA fork head-on and becomes backtracked, an RNA-DNA hybrid can form on the lagging strand in front of Pol II, creating a topological lock that traps Pol II at the fork. TFIIS facilitates RNA-DNA hybrid removal by severing the connection of Pol II with the hybrid. We further demonstrate that this RNA-DNA hybrid can prime lagging strand replication by T7 DNA polymerase while Pol II is still bound to DNA. Our findings capture basal properties of the interactions of Pol II with a DNA fork, revealing significant implications for transcription-replication conflicts.

biophysics↗

bioRxiv2024

Characterization of bacterial intrinsic transcription terminators identified with TERMITe - a novel method for comprehensive analysis of Term-seq data

In recent years, Term-seq became a standard experimental approach for high-throughput identification of 3 ends of bacterial transcripts. It was widely adopted to study transcription termination events and 3 maturation of bacterial RNAs. Despite widespread utilization, a universal bioinformatics toolkit for comprehensive analysis of Term-seq sequencing data is still lacking. Here, we describe TERMITe, a novel method for the identification of stable 3 RNA ends based on bacterial Term-seq data. TERMITe works with data obtained from both currently available Term-seq protocols and provides robust identification of the 3 RNA termini. Unique features of TERMITe include the calculation of the transcription termination efficiency using matched RNA-seq data and the comprehensive annotation of the identified 3 RNA ends, allowing functional analysis of the results. We have applied TERMITe to the comparative analysis of experimentally validated intrinsic terminators spanning different species across the bacterial domain of life, revealing substantial differences in their sequence and secondary structure. We also provide a complete atlas of experimentally validated intrinsic transcription termination sites for 13 bacterial species, including Escherichia coli, Bacillus subtilis, Listeria monocytogenes, Enterococcus faecalis, Synechocystis sp., Streptomyces clavuligerus, Streptomyces griseus, Streptomyces coelicolor, Streptomyces avermitilis, Streptomyces lividans, Streptomyces tsukubaensis, Streptomyces venezuelae, and Zymomonas mobilis.

bioinformatics↗

bioRxiv2022

The fidelity of transcription in human cells

Accurate transcription is required for the faithful expression of genetic information. To provide insight into the molecular mechanisms that control the fidelity of transcription, we analyzed the landscape of transcription errors in human embryonic stem cells. These measurements provide the first reasonable estimate of the fidelity of transcription in human cells and identify multiple genetic and epigenetic factors that control its accuracy. In addition, we developed a new reporter mouse to identify cell types and tissues that commit these errors the most. These experiments revealed that CA1 and dentate gyrus neurons are highly sensitive to transcriptional mutagenesis, lending new support to the hypothesis that transcription errors play a role in the progression of Alzheimers disease. Taken together, these experiments provide unprecedented insight into the fidelity of gene expression in human cells and the molecular mechanisms that govern the central dogma of life.

genetics↗

bioRxiv2020

Nascent RNA sequencing identifies a widespread sigma70-dependent pausing regulated by Gre factors in bacteria

Promoter-proximal pausing regulates expression of many eukaryotic genes and serves as checkpoints for assembly of elongation/splicing machinery. Little is known how broadly the pausing is employed in transcriptional regulation in bacteria. We applied NET-seq combined with RNase I footprinting for genome-wide analysis of {sigma}70-dependent transcription pauses in Escherichia coli. Many E. coli genes appear to contain clusters of strong backtracked pauses at 10-20-bp distance from the transcription start site caused by retention of {sigma}70 subunit in RNA polymerase. The pauses in 10-15-bp register of the promoter are dictated by binding of {sigma}70 to canonical -10 element, 6-7 nt spacer and "YR+1Y" motif centered at transcription start site all characteristic for strong E. coli promoters. The promoters for the pauses in 16-20-bp register contain an additional -10-like sequence positioned on the same face of the DNA duplex as the original -10 element suggesting that {sigma}70 hopping was responsible for these pauses. Our in vitro analysis reveals that RNA polymerase backtracking and DNA scrunching are involved in these pauses that are relieved by Gre transcript cleavage factors. The genes coding for transcription factors are enriched in these pauses suggesting that {sigma}70 and Gre proteins regulate transcription in response to changing environmental cues.

microbiology↗

bioRxiv2020

NusG is an intrinsic transcription termination factor that stimulates motility and coordinates global gene expression with NusA

NusA and NusG are transcription elongation factors that stimulate RNA polymerase pausing in Bacillus subtilis. While NusA was known to function as an intrinsic termination factor, the role of NusG in this process had not been explored. To examine the individual and combinatorial roles that NusA and NusG play in intrinsic termination, Term-seq was conducted in wild type, NusA depletion, {Delta}nusG, and NusA depletion {Delta}nusG strains. We determined that NusG functions as an intrinsic termination factor that works alone and cooperatively with NusA to facilitate termination at 88% of the 1,400 identified intrinsic terminators. The loss of both proteins leads to global misregulation of gene expression. Our results indicate that NusG stimulates a sequence-specific pause that assists in the completion of suboptimal terminator hairpins with weak terminal A-U and G-U base pairs at the bottom of the stem. Moreover, the loss of NusG results in flagella and swimming motility defects.

microbiology↗

bioRxiv2020

Escherichia coli σ38 promoters use two UP elements instead of a -35 element: resolution of a paradoxand discovery that σ38 transcribes ribosomal promoters

1In E. coli, one RNA polymerase (RNAP) transcribes all RNA species, and different regulons are transcribed by employing different sigma ({sigma}) factors. RNAP containing{sigma} 38 ({sigma}S) activates genes responding to stress conditions such as stationary phase. The structure of{sigma} 38 promoters has been controversial for more than two decades. To construct a model of{sigma} 38 promoters using information theory, we aligned proven transcriptional start sites to maximize the sequence information, in bits, and identified a -10 element similar to{sigma} 70 promoters. We could not align any -35 sequence logo; instead we found two patterns upstream of the -35 region. These patterns have dyad symmetry sequences and correspond to the location of UP elements in ribosomal RNA (rRNA) promoters. Additionally the UP element dyad symmetry suggests that the two polymerase subunits, which bind to the UPs, should have two-fold dyad axis of symmetry on the polymerase and this is indeed observed in an X-ray crystal structure. Curiously the CTDs should compete for overlapping UP elements. In vitro experiments confirm that{sigma} 38 recognizes the rrnB P1 promoter, requires a -10, UP elements and no -35. This clarifies the long-standing paradox of how{sigma} 38 promoters differ from those of{sigma} 70.

molecular biology↗