Regulation of Transcriptional Elongation in Escherichia Coli PDF Download
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Author: Rachel Anne Mooney
Publisher:
ISBN:
Category :
Languages : en
Pages : 302
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Author: Rachel Anne Mooney
Publisher:
ISBN:
Category :
Languages : en
Pages : 302
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Book Description
Author: E. C. C. Lin
Publisher: Springer Science & Business Media
ISBN: 1468486012
Category : Medical
Languages : en
Pages : 1010
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Book Description
This up-to-date guide focuses on the understanding of key regulatory mechanisms governing gene expression in Escherichia coli. Studies of E. coli not only provide the first models of gene regulation, but research continues to yield different control mechanisms.
Author: Chuanhai Zheng
Publisher:
ISBN:
Category :
Languages : en
Pages : 264
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Author: Yu Bao (Ph.D.)
Publisher:
ISBN:
Category :
Languages : en
Pages : 0
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Gene expression in all life forms relies on multi-subunit RNA polymerase (RNAP). In transcription elongation stage, RNAP senses specific DNA signals and enters an offline, pausing state, which has multiple interconverting subtypes and allows versatile regulatory options. This work focuses on understanding the pausing regulatory function of sequence insertion 3 (SI3) in Escherichia coli (E. coli) RNAP. In E. coli, the catalytic trigger loop (TL) is interrupted by surface-exposed, 188-aa sequence insertion 3 (SI3). SI3 occupies different locations in paused transcription complexes, but its dynamics during pausing are undefined. We report design, optimization, and use of a Cys-triplet reporter to measure the positional bias of SI3 in different transcription complexes and to determine the effect of restricting SI3 movement on pausing. RNA hairpin-stabilized pausing biases SI3 toward a swiveled position that inhibits TL folding while the consensus elemental pausing biases SI3 toward a closed position that potentially stabilized a pre-translocated state of RNAP. Our findings establish that SI3 functions by modulating the TL folding to aid transcriptional regulation. To investigate SI3's genomic function in transcriptional pausing, we constructed a viable E. coli strain lacking SI3 enabled by a suppressor TL substitution ([beta]'Ala941-Thr; [delta]SI3*). [delta]SI3* increased transcription rate in vitro relative to [delta]SI3, possibly explaining its viability, but retained both positive and negative effects of [delta]SI3 on pausing. Using NET-seq, we found that [delta]SI3*-enhanced pauses resemble the consensus elemental pause sequence whereas sequences at [delta]SI3*-suppressed pauses, which exhibited greater association with PHs, were more divergent. [delta]SI3*-suppressed pauses also were associated with apparent pausing one nt upstream from the consensus sequence, often generating tandem pause sites. Our results document multiple ways that SI3 modulates pausing in vivo and may explain differences in consensus pause sequences found in some NET-seq studies. In conclusion, our comprehensive studies provide compelling evidence linking the movements of the SI3 domain to the formation and stabilization of diverse transcriptional pause signals. We demonstrate the potential of biochemical crosslink assays in exploring conformational changes within large protein complexes. Additionally, our research emphasizes the necessity for refining current NET-seq protocols to prevent unintended cleavage during library preparation.
Author: Ole Maaløe
Publisher:
ISBN:
Category : Bacteria
Languages : en
Pages : 306
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Author: Asma Issam Hatoum
Publisher:
ISBN:
Category :
Languages : en
Pages : 302
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Author: Melanie Marie Barker
Publisher:
ISBN:
Category :
Languages : en
Pages : 354
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Author: Markus Willard Covert
Publisher:
ISBN:
Category :
Languages : en
Pages : 434
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Author: Susan Marie Uptain
Publisher:
ISBN:
Category :
Languages : en
Pages : 604
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Author: Smarajit Mondal
Publisher:
ISBN:
Category :
Languages : en
Pages :
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Transcription in bacteria is regulated at the level of initiation, elongation and termination. Although the regulation of transcriptional initiation is well studied, the regulation of elongation and termination are not well understood. This thesis focuses on understanding the role of NusA on intrinsic termination and the role of NusG on RNA polymerase pausing using genomic, biochemical and computational analyses. Tight regulation of transcription termination is required to maintain proper levels of gene expression in bacteria, because termination failure abolishes operon boundaries, leading to misregulation of downstream genes. NusA is a negative transcription elongation factor that was known to cause a slight stimulation of termination at intrinsic terminators in vitro, but its impact on termination and global gene expression in vivo was not known. In this thesis, I describe the mapping of intrinsic terminators genome wide in B subtilis and measure the effect of NusA on the efficiency of these terminators in vivo using a novel high resolution 3' end-mapping technique coupled with mRNA profiling. Based on these studies, I report the existence of a subclass of previously unidentified pseudo-intrinsic terminators that are dependent on NusA for termination. Sequence comparison of different terminators reveals that weak hairpins and/or distal U-tract interruptions favors NusA-dependent termination, supporting a model in which NusA assists hairpin folding and slows down RNA polymerase near the termination window. These studies also revealed that readthrough of NusA-dependent terminators increases transcription of genes related to replication and DNA metabolism, suggesting a role of NusA in maintaining genome stability. I further show that nusA is autoregulated by a transcription attenuation mechanism that does not rely on antiterminator structures to prevent termination. Instead, NusA-stimulated termination in its 5'UTR dictates the extent of transcription into the operon. Another major focus of this thesis is to understand the regulation of transcription elongation by NusG-stimulated pausing of RNA polymerase. NusG is a positive elongation factor in E. coli that accelerates transcription by reducing the dwell time of RNA polymerase at pause sites. In B. subtilis, NusG stimulates pausing at positions U107 and U144 in the trp-leader transcript. NusG-stimulated pausing at U144 requires a short sequence in the non-template DNA strand and participates in the TRAP-dependent translation repression mechanism. In this thesis, I report the characterization of the NusG-stimulated U107 pause signal and show that disruption of the NusG recognition motif dramatically reduces pausing. These results suggest a mechanism in which RNA polymerase pausing at this site participates in the transcription attenuation mechanism by increasing additional time for TRAP binding to the nascent transcript.
