scholarly journals Escherichia coli NusG links the lead ribosome with the transcription elongation complex

2019 ◽  
Author(s):  
Robert S. Washburn ◽  
Philipp K. Zuber ◽  
Ming Sun ◽  
Yaser Hashem ◽  
Bingxin Shen ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Conclusive Evidence ◽  
Resonance Spectroscopy ◽  
Elongation Complex ◽  
Magnetic Resonance Spectroscopy Data ◽  
N Utilization ◽  
Reporter Assays ◽  
70S Ribosome ◽  
Functional Analyses

AbstractIt has been known for more than 50 years that transcription and translation are physically coupled in bacteria, but whether or not this coupling may be mediated by the two-domain protein N-utilization substance (Nus) G in Escherichia coli is still heavily debated. Here, we combine integrative structural biology and functional analyses to provide conclusive evidence that NusG can physically link transcription with translation by contacting both RNA polymerase and the ribosome. We present a cryo-electron microscopy structure of a NusG:70S ribosome complex and nuclear magnetic resonance spectroscopy data revealing simultaneous binding of NusG to RNAP and the intact 70S ribosome, providing the first direct structural evidence for NusG-mediated coupling. Furthermore, in vivo reporter assays show that recruitment of NusG occurs late in transcription and strongly depends on translation. Thus, our data suggest that coupling occurs initially via direct RNAP:ribosome contacts and is then mediated by NusG.

scholarly journals Poor Mitochondrial Health and Systemic Inflammation? Testing of a Classical Hypothesis

2020 ◽  
Vol 4 (Supplement_1) ◽  
pp. 126-127
Author(s):  
Marta Zampino ◽  
Luigi Ferrucci ◽  
Richard Spencer ◽  
Kenneth Fishbein ◽  
Eleanor Simonsick ◽  
...  
Keyword(s):  
Mitochondrial Function ◽  
Fat Body ◽  
Reference Group ◽  
Linear Regression Analysis ◽  
Oxidative Capacity ◽  
Conclusive Evidence ◽  
Resonance Spectroscopy ◽  
Low Grade ◽  
Severe Impairment

Abstract Chronic low-grade inflammation often occurs with aging and has been associated with negative health outcomes. Despite extensive research on the origins of “inflammaging”, the causative mechanisms remain unclear. However, a connection between poor mitochondrial health and chronic inflammation has been hypothesized, with decreasing mitochondrial function occurring with age and precipitating an increase in reactive oxygen species and other pro-inflammatory macromolecules such as mitochondrial DNA. We tested this hypothesis on a population of 619 subjects from the Baltimore Longitudinal Study of Aging, measuring muscle mitochondrial oxidative capacity in vivo by phosphorus magnetic resonance spectroscopy (P-MRS), and plasma interleukin (IL)-6, the most widely used biomarker of inflammaging. The P-MRS-derived post-exercise phosphocreatine recovery time constant tau-PCr, a measure of oxidative capacity, was expressed as a categorical variable through assignment to quintiles. Participants in the first quintile of tau-PCr (best mitochondrial function) were taken as reference and compared to the others using linear regression analysis adjusted for sex, age, lean and fat body mass, and physical activity. Those participants with the lowest oxidative capacity had significantly higher log(IL-6) levels as compared to the reference group. However, data from the other quintiles was not significantly different from the reference values. In conclusion, severe impairment of oxidative capacity is associated with increased inflammation. This study design does not provide conclusive evidence of whether increased inflammation and impaired bioenergetic recovery are both caused by underlying poor health status, or whether mitochondrial deficits lead directly to the observed inflammation; we anticipate addressing this important question with longitudinal studies.

scholarly journals Genetic and Functional Analyses of the Conserved C-Terminal Core Domain of Escherichia coli FtsZ

1999 ◽  
Vol 181 (24) ◽  
pp. 7531-7544 ◽  
Author(s):  
Xiaolan Ma ◽  
William Margolin
Keyword(s):  
Escherichia Coli ◽  
Amino Acid ◽  
Immunoblot Analysis ◽  
Conserved Residues ◽  
Core Domain ◽  
Mutant Proteins ◽  
E Coli ◽  
Stable Mutant ◽  
Functional Analyses

ABSTRACT In Escherichia coli, FtsZ is required for the recruitment of the essential cell division proteins FtsA and ZipA to the septal ring. Several C-terminal deletions of E. coliFtsZ, including one of only 12 amino acids that removes the highly conserved C-terminal core domain, failed to complement chromosomalftsZ mutants when expressed on a plasmid. To identify key individual residues within the core domain, six highly conserved residues were replaced with alanines. All but one of these mutants (D373A) failed to complement an ftsZ chromosomal mutant. Immunoblot analysis demonstrated that whereas I374A and F377A proteins were unstable in the cell, L372A, D373A, P375A, and L378A proteins were synthesized at normal levels, suggesting that they were specifically defective in some aspect of FtsZ function. In addition, all four of the stable mutant proteins were able to localize and form rings at potential division sites in chromosomal ftsZ mutants, implying a defect in a function other than localization and multimerization. Because another proposed function of FtsZ is the recruitment of FtsA and ZipA, we tested whether the C-terminal core domain was important for interactions with these proteins. Using two different in vivo assays, we found that the 12-amino-acid truncation of FtsZ was defective in binding to FtsA. Furthermore, two point mutants in this region (L372A and P375A) showed weakened binding to FtsA. In contrast, ZipA was capable of binding to all four stable point mutants in the FtsZ C-terminal core but not to the 12-amino-acid deletion.

scholarly journals Highly Divergent RfaH Orthologs from Pathogenic Proteobacteria Can Substitute for Escherichia coli RfaH both In Vivo and In Vitro

2004 ◽  
Vol 186 (9) ◽  
pp. 2829-2840 ◽  
Author(s):  
Heather D. Carter ◽  
Vladimir Svetlov ◽  
Irina Artsimovitch
Keyword(s):  
Escherichia Coli ◽  
Expression Vectors ◽  
Divergent Evolution ◽  
Transcriptional Enhancer ◽  
Elongation Complex ◽  
E Coli ◽  
Serovar Typhimurium ◽  
Transcript Elongation

ABSTRACT The transcriptional enhancer protein RfaH positively regulates production of virulence factors in Escherichia coli and Salmonella enterica serovar Typhimurium via a cis element, ops. Genes coding for RfaH orthologs were identified in conceptually translated genomes of bacterial pathogens, including Vibrio and Yersinia spp. We cloned the rfaH genes from Vibrio cholerae, Yersinia enterocolitica, S. enterica serovar Typhimurium, and Klebsiella pneumoniae into E. coli expression vectors. Purified RfaH orthologs, including the most divergent one from V. cholerae, were readily recruited to the E. coli transcription elongation complex. Postrecruitment stimulation of transcript elongation appeared to vary with the degree of similarity to E. coli RfaH. V. cholerae RfaH was particularly defective in reducing downstream pausing and termination; this defect was substantially alleviated by an increase in its concentration. When overexpressed episomally, all of the rfaH genes complemented the disruption of the chromosomal copy of the E. coli gene. Thus, despite the apparently accelerated divergent evolution of the RfaH proteins, the mechanism of their action is conserved well enough to make them transcriptionally active in the E. coli system.

scholarly journals The primary σ factor in Escherichia coli can access the transcription elongation complex from solution in vivo

eLife ◽  
2015 ◽  
Vol 4 ◽  
Author(s):  
Seth R Goldman ◽  
Nikhil U Nair ◽  
Christopher D Wells ◽  
Bryce E Nickels ◽  
Ann Hochschild
Keyword(s):  
Escherichia Coli ◽  
Transcription Elongation ◽  
Elongation Factor ◽  
Direct Effects ◽  
Elongation Complex ◽  
Initiation Factors ◽  
Bacterial Rna ◽  
Σ Factor ◽  
In Trans

The σ subunit of bacterial RNA polymerase (RNAP) confers on the enzyme the ability to initiate promoter-specific transcription. Although σ factors are generally classified as initiation factors, σ can also remain associated with, and modulate the behavior of, RNAP during elongation. Here we establish that the primary σ factor in Escherichia coli, σ70, can function as an elongation factor in vivo by loading directly onto the transcription elongation complex (TEC) in trans. We demonstrate that σ70 can bind in trans to TECs that emanate from either a σ70-dependent promoter or a promoter that is controlled by an alternative σ factor. We further demonstrate that binding of σ70 to the TEC in trans can have a particularly large impact on the dynamics of transcription elongation during stationary phase. Our findings establish a mechanism whereby the primary σ factor can exert direct effects on the composition of the entire transcriptome, not just that portion that is produced under the control of σ70-dependent promoters.

Functional Analyses of Escherichia coli MutS-β Clamp Interaction In Vitro and In Vivo

2009 ◽  
Vol 60 (6) ◽  
pp. 466-470 ◽  
Author(s):  
Ying Zhou ◽  
Feng Li ◽  
Yuanyuan Chen ◽  
Lijun Bi ◽  
Xian-En Zhang
Keyword(s):  
Escherichia Coli ◽  
Functional Analyses

scholarly journals Nuclear magnetic resonance spectroscopy reveals the functional state of the signalling protein CheY in vivo in Escherichia coli

2003 ◽  
Vol 49 (5) ◽  
pp. 1191-1200 ◽  
Author(s):  
Julia A. Hubbard ◽  
Lesley K. MacLachlan ◽  
Gavin W. King ◽  
Joanna J. Jones ◽  
Andrew P. Fosberry
Keyword(s):  
Escherichia Coli ◽  
Nuclear Magnetic Resonance ◽  
Magnetic Resonance ◽  
Magnetic Resonance Spectroscopy ◽  
Nuclear Magnetic Resonance Spectroscopy ◽  
Functional State ◽  
Resonance Spectroscopy ◽  
Nuclear Magnetic
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