scholarly journals Accounting for RNA polymerase heterogeneity reveals state switching and two distinct long-lived backtrack states escaping through cleavage

2019 ◽  
Author(s):  
Richard Janissen ◽  
Behrouz Eslami-Mossallam ◽  
Irina Artsimovitch ◽  
Martin Depken ◽  
Nynke H. Dekker
Keyword(s):  
Rna Polymerase ◽  
Brownian Dynamics ◽  
Mechanistic Model ◽  
Magnetic Tweezers ◽  
Rna Cleavage ◽  
E Coli ◽  
Bacterial Rna ◽  
Wide Range ◽  
Structural Isomerization ◽  
Stochastic Sources

ABSTRACTPausing by bacterial RNA polymerase (RNAp) is vital in the recruitment of regulatory factors, RNA folding, and coupled translation. While backtracking and intra-structural isomerization have been proposed to trigger pausing, our understanding of backtrack-associated pauses and catalytic recovery remains incomplete. Using high-throughput magnetic tweezers, we examined the E. coli RNAp transcription dynamics over a wide range of forces and NTP concentrations. Dwell-time analysis and stochastic modeling identified, in addition to a short-lived elemental pause, two distinct long-lived backtrack pause states differing in recovery rates. We further identified two stochastic sources of transcription heterogeneity: alterations in short-pause frequency that underlie elongation-rate switching, and RNA cleavage deficiency that underpins different long-lived backtrack states. Together with effects of force and Gre factors, we demonstrate that recovery from deep backtracks is governed by intrinsic RNA cleavage rather than diffusional Brownian dynamics. We introduce a consensus mechanistic model that unifies our findings with prior models.

scholarly journals Identification and characterization of pleiotropic high-persistence mutations in the beta subunit of the bacterial RNA polymerase

2021 ◽  
Author(s):  
Lev Ostrer ◽  
Yinduo Ji ◽  
Arkady Khodursky
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Iii ◽  
Survival Rates ◽  
Beta Subunit ◽  
High Survival ◽  
Resistance Mutations ◽  
Switch Region ◽  
E Coli ◽  
Bacterial Rna ◽  
Survival Frequency

Mutations conferring resistance to bactericidal antibiotics reduce average susceptibility of the mutant populations. It is unknown, however, how those mutations affect survival of individual bacteria. Since surviving bacteria can be a reservoir for recurring infections, it is important to know how survival rates may be affected by resistance mutations and by the choice of antibiotics. Here we present the evidence that: i) Escherichia coli mutants with 100-1000 times increased frequency of survival in ciprofloxacin, an archetypal fluoroquinolone antibiotic, can be readily obtained in a stepwise selection; ii) the high survival frequency is conferred by mutations in the switch region of the beta subunit of the RNA polymerase; iii) the switch-region mutations are (p)ppGpp mimics, partially analogous to rpoB stringent mutations; iv) the stringent and switch-region rpoB mutations frequently occur in clinical isolates of E. coli , Acinetobacter baumannii , Mycobacterium tuberculosis and Staphylococcus aureus , and at least one of them, RpoB S488L, which is a common rifampicin-resistance mutations, dramatically increases survival of a clinical MRSA strain in ampicillin; v) the RpoB-associated high-survival phenotype can be reversed by sub-inhibitory concentrations of chloramphenicol.

INTRAGENIC PROMOTOR-LIKE SITES IN THE GENOME OFESCHERICHIA COLIDISCOVERY AND FUNCTIONAL IMPLICATION

2007 ◽  
Vol 05 (02b) ◽  
pp. 549-560 ◽  
Author(s):  
MARIA N. TUTUKINA ◽  
KONSTANTIN S. SHAVKUNOV ◽  
IRINA S. MASULIS ◽  
OLGA N. OZOLINE
Keyword(s):  
Rna Polymerase ◽  
Antisense Transcription ◽  
Antisense Strand ◽  
Entire Genome ◽  
Coding Sequences ◽  
E Coli ◽  
Chip Technology ◽  
Bacterial Rna ◽  
Small Regulatory Rnas ◽  
Polymerase Binding

Mapping of putative promoters within the entire genome of Escherichia coli (E. coli) by means of pattern-recognition software PlatProm revealed several thousand of sites having high probability to perform promoter function. Along with the expected promoters located upstream of coding sequences, PlatProm identified more than a thousand potential promoters for antisense transcription and several hundred very similar signals within coding sequences having the same direction with the genes. Since recently developed ChIP–chip technology also testified the presence of intragenic RNA polymerase binding sites, such distribution of putative promoters is likely to be a general biological phenomenon reflecting yet undiscovered regulatory events. Here, we provide experimental evidences that two internal promoters are recognized by bacterial RNA polymerase. One of them is located within the hns coding sequence and may initiate synthesis of RNA from the antisense strand. Another one is found within the overlapping genes htgA/yaaW and may control the production of a shortened mRNA or an RNA-product complementary to mRNA of yaaW. Both RNA-products can form secondary structures with free energies of folding close to those of small regulatory RNAs (sRNAs) of the same length. Folding propensity of known sRNAs was further compared with that of antisense RNAs (aRNAs), predicted in E. coli as well as in Salmonella typhimurium (S. typhimurium). Slightly lower stability observed for aRNAs assumes that their structural compactness may be less significant for biological function.

scholarly journals A T7 phage factor required for managing RpoS inEscherichia coli

2018 ◽  
Author(s):  
Aline Tabib-Salazar ◽  
Bing Liu ◽  
Declan Barker ◽  
Lynn Burchell ◽  
Udi Qimron ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Rna Polymerase ◽  
Stationary Phase ◽  
Stationary Phases ◽  
Specific Inhibitor ◽  
E Coli ◽  
Bacterial Transcription ◽  
Bacterial Rna ◽  
Phage T7 ◽  
T7 Phage

T7 development inEscherichia colirequires the inhibition of the housekeeping form of the bacterial RNA polymerase (RNAP), Eσ70, by two T7 proteins: Gp2 and Gp5.7. While the biological role of Gp2 is well understood, that of Gp5.7 remains to be fully deciphered. Here, we present results from functional and structural analyses to reveal that Gp5.7 primarily serves to inhibit EσS, the predominant form of the RNAP in the stationary phase of growth, which accumulates in exponentially growingE. colias a consequence of buildup of guanosine pentaphosphate ((p)ppGpp) during T7 development. We further demonstrate a requirement of Gp5.7 for T7 development inE. colicells in the stationary phase of growth. Our finding represents a paradigm for how some lytic phages have evolved distinct mechanisms to inhibit the bacterial transcription machinery to facilitate phage development in bacteria in the exponential and stationary phases of growth.Significance statementVirus that infect bacteria (phages) represent the most abundant living entities on the planet and many aspects of our fundamental knowledge of phage-bacteria relationships have been derived in the context of exponentially growing bacteria. In the case of the prototypicalEscherichia coliphage T7, specific inhibition of the housekeeping form of the RNA polymerase (Eσ70) by a T7 protein, called Gp2, is essential for the development of viral progeny. We now reveal that T7 uses a second specific inhibitor that selectively inhibits the stationary phase RNAP (EσS), which enables T7 to develop well in exponentially growing and stationary phase bacteria. The results have broad implications for our understanding of phage-bacteria relationships and therapeutic application of phages.

scholarly journals Recognition of Streptococcal Promoters by the Pneumococcal SigA Protein

2021 ◽  
Vol 8 ◽  
Author(s):  
Virtu Solano-Collado ◽  
Sofía Ruiz-Cruz ◽  
Fabián Lorenzo-Díaz ◽  
Radoslaw Pluta ◽  
Manuel Espinosa ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Sigma Factor ◽  
Regulation Of Gene Expression ◽  
E Coli ◽  
Core Enzyme ◽  
Protein Architecture ◽  
Bacterial Rna ◽  
Promoter Recognition ◽  
Rna Polymerase Holoenzyme

Promoter recognition by RNA polymerase is a key step in the regulation of gene expression. The bacterial RNA polymerase core enzyme is a complex of five subunits that interacts transitory with one of a set of sigma factors forming the RNA polymerase holoenzyme. The sigma factor confers promoter specificity to the RNA polymerase. In the Gram-positive pathogenic bacterium Streptococcus pneumoniae, most promoters are likely recognized by SigA, a poorly studied housekeeping sigma factor. Here we present a sequence conservation analysis and show that SigA has similar protein architecture to Escherichia coli and Bacillus subtilis homologs, namely the poorly conserved N-terminal 100 residues and well-conserved rest of the protein (domains 2, 3, and 4). Further, we have purified the native (untagged) SigA protein encoded by the pneumococcal R6 strain and reconstituted an RNA polymerase holoenzyme composed of the E. coli core enzyme and the sigma factor SigA (RNAP-SigA). By in vitro transcription, we have found that RNAP-SigA was able to recognize particular promoters, not only from the pneumococcal chromosome but also from the S. agalactiae promiscuous antibiotic-resistance plasmid pMV158. Specifically, SigA was able to direct the RNA polymerase to transcribe genes involved in replication and conjugative mobilization of plasmid pMV158. Our results point to the versatility of SigA in promoter recognition and its contribution to the promiscuity of plasmid pMV158.

scholarly journals Validation of Omega Subunit of RNA Polymerase as a Functional Entity

Biomolecules ◽  
2020 ◽  
Vol 10 (11) ◽  
pp. 1588
Author(s):  
Unnatiben Rajeshbhai Patel ◽  
Sudhanshu Gautam ◽  
Dipankar Chatterji
Keyword(s):  
Rna Polymerase ◽  
Structural Alteration ◽  
Lethal Mutant ◽  
Catalytic Center ◽  
Reading Frame ◽  
E Coli ◽  
Dominant Lethal ◽  
Bacterial Rna ◽  
Considerable Period

The bacterial RNA polymerase (RNAP) is a multi-subunit protein complex (α2ββ’ω σ) containing the smallest subunit, ω. Although identified early in RNAP research, its function remained ambiguous and shrouded with controversy for a considerable period. It was shown before that the protein has a structural role in maintaining the conformation of the largest subunit, β’, and its recruitment in the enzyme assembly. Despite evolutionary conservation of ω and its role in the assembly of RNAP, E. coli mutants lacking rpoZ (codes for ω) are viable due to the association of the global chaperone protein GroEL with RNAP. To get a better insight into the structure and functional role of ω during transcription, several dominant lethal mutants of ω were isolated. The mutants showed higher binding affinity compared to that of native ω to the α2ββ’ subassembly. We observed that the interaction between α2ββ’ and these lethal mutants is driven by mostly favorable enthalpy and a small but unfavorable negative entropy term. However, during the isolation of these mutants we isolated a silent mutant serendipitously, which showed a lethal phenotype. Silent mutant of a given protein is defined as a protein having the same sequence of amino acids as that of wild type but having mutation in the gene with alteration in base sequence from more frequent code to less frequent one due to codon degeneracy. Eventually, many silent mutants were generated to understand the role of rare codons at various positions in rpoZ. We observed that the dominant lethal mutants of ω having either point mutation or silent in nature are more structured in comparison to the native ω. However, the silent code’s position in the reading frame of rpoZ plays a role in the structural alteration of the translated protein. This structural alteration in ω makes it more rigid, which affects the plasticity of the interacting domain formed by ω and α2ββ’. Here, we attempted to describe how the conformational flexibility of the ω helps in maintaining the plasticity of the active site of RNA polymerase. The dominant lethal mutant of ω has a suppressor mapped near the catalytic center of the β’ subunit, and it is the same for both types of mutants.

RNA polymerase: Preparation and structural analysis of helical polymers

1984 ◽  
Vol 42 ◽  
pp. 152-153
Author(s):  
E. Loren Buhle ◽  
Pamela Rew ◽  
Ueli Aebi
Keyword(s):  
Structural Analysis ◽  
Rna Polymerase ◽  
Molecular Level ◽  
X Ray Diffraction ◽  
Helical Polymers ◽  
X Ray ◽  
E Coli ◽  
The Past ◽  
Ordered Arrays ◽  
Low Ionic Strength

While DNA-dependent RNA polymerase represents one of the key enzymes involved in transcription and ultimately in gene expression in procaryotic and eucaryotic cells, little progress has been made towards elucidation of its 3-D structure at the molecular level over the past few years. This is mainly because to date no 3-D crystals suitable for X-ray diffraction analysis have been obtained with this rather large (MW ~500 kd) multi-subunit (α2ββ'ζ). As an alternative, we have been trying to form ordered arrays of RNA polymerase from E. coli suitable for structural analysis in the electron microscope combined with image processing. Here we report about helical polymers induced from holoenzyme (α2ββ'ζ) at low ionic strength with 5-7 mM MnCl2 (see Fig. 1a). The presence of the ζ-subunit (MW 86 kd) is required to form these polymers, since the core enzyme (α2ββ') does fail to assemble into such structures under these conditions.

Small Molecule Permeation Across Membrane Channels: Chemical Modification to Quantify Transport Across OmpF

2018 ◽  
Author(s):  
Jiajun Wang ◽  
Jayesh Arun Bafna ◽  
Satya Prathyusha Bhamidimarri ◽  
Mathias Winterhalter
Keyword(s):  
Dwell Time ◽  
Covalent Binding ◽  
Channel Structure ◽  
Ion Current ◽  
E Coli ◽  
Current Fluctuation ◽  
Wide Range ◽  
Outer Membrane Channel ◽  
Biological Channels ◽  
Constriction Region

Biological channels facilitate the exchange of small molecules across membranes, but surprisingly there is a lack of general tools for the identification and quantification of transport (i.e., translocation and binding). Analyzing the ion current fluctuation of a typical channel with its constriction region in the middle does not allow a direct conclusion on successful transport. For this, we created an additional barrier acting as a molecular counter at the exit of the channel. To identify permeation, we mainly read the molecule residence time in the channel lumen as the indicator whether the molecule reached the exit of the channel. As an example, here we use the well-studied porin, OmpF, an outer membrane channel from <i>E. coli</i>. Inspection of the channel structure suggests that aspartic acid at position 181 is located below the constriction region (CR) and we subsequently mutated this residue to cysteine, where else cysteine free and functionalized it by covalent binding with 2-sulfonatoethyl methanethiosulfonate (MTSES) or the larger glutathione (GLT) blockers. Using the dwell time as the signal for transport, we found that both mono-arginine and tri-arginine permeation process is prolonged by 20% and 50% respectively through OmpF<sub>E181C</sub>MTSES, while the larger sized blocker modification OmpF<sub>E181C</sub>GLT drastically decreased the permeation of mono-arginine by 9-fold and even blocked the pathway of the tri-arginine. In case of the hepta-arginine as substrate, both chemical modifications led to an identical ‘blocked’ pattern observed by the dwell time of ion current fluctuation of the OmpF<sub>wt</sub>. As an instance for antibiotic permeation, we analyzed norfloxacin, a fluoroquinolone antimicrobial agent. The modulation of the interaction dwell time suggests possible successful permeation of norfloxacin across OmpF<sub>wt</sub>. This approach may discriminate blockages from translocation events for a wide range of substrates. A potential application could be screening for scaffolds to improve the permeability of antibiotics.

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