scholarly journals Suppression of ΔbipA Phenotypes in Escherichia coli by Abolishment of Pseudouridylation at Specific Sites on the 23S rRNA

2008 ◽  
Vol 190 (23) ◽  
pp. 7675-7683 ◽  
Author(s):  
Karthik Krishnan ◽  
Ann M. Flower
Keyword(s):  
Escherichia Coli ◽  
Target Genes ◽  
Regulatory Protein ◽  
Elongation Factor ◽  
Direct Interaction ◽  
Cold Sensitivity ◽  
23S Rrna ◽  
Pseudouridine Synthase ◽  
Suppressor Effect ◽  
Cold Sensitive

ABSTRACT The BipA protein of Escherichia coli has intriguing similarities to the elongation factor subfamily of GTPases, including EF-Tu, EF-G, and LepA. In addition, phenotypes of a bipA deletion mutant suggest that BipA is involved in regulation of a variety of pathways. These two points have led to speculation that BipA may be a novel regulatory protein that affects efficient translation of target genes through direct interaction with the ribosome. We isolated and characterized suppressors of the cold-sensitive growth phenotype exhibited by ΔbipA strains and identified insertion mutations in rluC. The rluC gene encodes a pseudouridine synthase responsible for pseudouridine modification of 23S rRNA at three sites, all located near the peptidyl transferase center. Deletion of rluC not only suppressed cold sensitivity but also alleviated the decrease in capsule synthesis exhibited by bipA mutants, suggesting that the phenotypic effects of BipA are manifested through an effect on the ribosome. The suppressor effect is specific to rluC, as deletion of other rlu genes did not relieve cold sensitivity, and further, more than a single pseudouridine residue is involved, as alteration of single residues did not produce suppressors. These results are consistent with a role for BipA in either the structure or the function of the ribosome and imply that wild-type ribosomes are dependent on BipA for efficient expression of target mRNAs and that the lack of pseudouridylation at these three sites renders the ribosomes BipA independent.

scholarly journals Efficient Assembly of Ribosomes Is Inhibited by Deletion ofbipAin Escherichia coli

2015 ◽  
Vol 197 (10) ◽  
pp. 1819-1827 ◽  
Author(s):  
Promisree Choudhury ◽  
Ann M. Flower
Keyword(s):  
Low Temperature ◽  
Modified Nucleosides ◽  
Cold Sensitivity ◽  
23S Rrna ◽  
Ribosome Assembly ◽  
Rrna Processing ◽  
Pseudouridine Synthase ◽  
Assembly Factor ◽  
Cold Sensitive ◽  
And Function

ABSTRACTThe bacterial BipA protein belongs to the EF-G family of translational GTPases and has been postulated to be either a regulatory translation factor or a ribosome assembly factor. To distinguish between these hypotheses, we analyzed the effect ofbipAdeletion on three phenotypes associated with ribosome assembly factors: cold sensitivity, ribosome subunit distribution, and rRNA processing. We demonstrated that a ΔbipAstrain exhibits a cold-sensitive phenotype that is similar to, and synergistic with, that of a strain with a known ribosome assembly factor,deaD. Additionally, thebipAdeletion strain displayed a perturbed ribosome subunit distribution when grown at low temperature, similar to that of adeaDmutant, and again, the double mutant showed additive effects. The primary ribosomal deficiency noted was a decreased level of the 50S subunit and the appearance of a presumed pre-50S particle. Finally, deletion ofbipAresulted in accumulation of pre23S rRNA, as did deletion ofdeaD. We further found that deletion ofrluC, which encodes a pseudouridine synthase that modifies the 23S rRNA at three sites, suppressed all three phenotypes of thebipAmutant, supporting and extending previous findings. Together, these results suggest that BipA is important for the correct and efficient assembly of the 50S subunit of the ribosome at low temperature but when unmodified by RluC, the ribosomes become BipA independent for assembly.IMPORTANCEThe ribosome is the complex ribonucleoprotein machine responsible for protein synthesis in all cells. Although much has been learned about the structure and function of the ribosome, we do not fully understand how it is assembled or the accessory proteins that increase efficiency of biogenesis and function. This study examined one such protein, BipA. Our results indicate that BipA either directly or indirectly enhances the formation of the 50S subunit of the ribosome, particularly at low temperature. In addition, ribosomes contain a large number of modified nucleosides, including pseudouridines. This work demonstrates that the function of BipA is tied to the modification status of the ribosome and may help us understand why these modifications have been retained.

scholarly journals Function of the Histone-Like Protein H-NS in Motility of Escherichia coli: Multiple Regulatory Roles Rather than Direct Action at the Flagellar Motor

2015 ◽  
Vol 197 (19) ◽  
pp. 3110-3120 ◽  
Author(s):  
Eun A Kim ◽  
David F. Blair
Keyword(s):  
Escherichia Coli ◽  
Direct Action ◽  
Regulatory Protein ◽  
Direct Interaction ◽  
Flagellar Motor ◽  
Flagellar Motility ◽  
Standard View ◽  
Content Type ◽  
Regulatory Pathways ◽  
Motor Interaction

ABSTRACTA number of investigations ofEscherichia colihave suggested that the DNA-binding protein H-NS, in addition to its well-known functions in chromosome organization and gene regulation, interacts directly with the flagellar motor to modulate its function. Here, in a study initially aimed at characterizing the H-NS/motor interaction further, we identify problems and limitations in the previous work that substantially weaken the case for a direct H-NS/motor interaction. Nullhnsmutants are immotile, largely owing to the downregulation of the flagellar master regulators FlhD and FlhC. We, and others, previously reported that anhnsmutant remains poorly motile even when FlhDC are expressed constitutively. In the present work, we use better-engineered strains to show that the motility defect in a Δhns, FlhDC-constitutive strain is milder than that reported previously and does not point to a direct action of H-NS at the motor. H-NS regulates numerous genes and might influence motility via a number of regulatory molecules besides FlhDC. To examine the sources of the motility defect that persists in an FlhDC-constitutive Δhnsmutant, we measured transcript levels and overexpression effects of a number of genes in candidate regulatory pathways. The results indicate that H-NS influences motility via multiple regulatory linkages that include, minimally, the messenger molecule cyclic di-GMP, the biofilm regulatory protein CsgD, and the sigma factors σSand σF. The results are in accordance with the more standard view of H-NS as a regulator of gene expression rather than a direct modulator of flagellar motor performance.IMPORTANCEData from a number of previous studies have been taken to indicate that the nucleoid-organizing protein H-NS influences motility not only by its well-known DNA-based mechanisms but also by binding directly to the flagellar motor to alter function. In this study, H-NS is shown to influence motility through diverse regulatory pathways, but a direct interaction with the motor is not supported. Previous indications of a direct action at the motor appear to be related to the use of nonnull strains and, in some cases, a failure to effectively bypass the requirement for H-NS in the expression of the flagellar regulon. These findings call for a substantially revised interpretation of the literature concerning H-NS and flagellar motility and highlight the importance of H-NS in diverse regulatory processes involved in the motile-sessile transition.

scholarly journals Persister Cells Resuscitate via Ribosome Modification by 23S rRNA Pseudouridine Synthase RluD

2019 ◽  
Author(s):  
Sooyeon Song ◽  
Thomas K. Wood
Keyword(s):  
Escherichia Coli ◽  
Small Rna ◽  
23S Rrna ◽  
Bacterial Cells ◽  
Persister Cells ◽  
E Coli ◽  
Pseudouridine Synthase ◽  
Dormant Cells ◽  
Wide Range ◽  
Persister Cell

ABSTRACTUpon a wide range of stress conditions (e.g., nutrient, antibiotic, oxidative), a subpopulation of bacterial cells known as persisters survive by halting metabolism. These cells resuscitate rapidly to reconstitute infections once the stress is removed and nutrients are provided. However, how these dormant cells resuscitate is not understood well but involves reactivating ribosomes. By screening 10,000 compounds directly for stimulating Escherichia coli persister cell resuscitation, we identified that 2-{[2-(4-bromophenyl)-2-oxoethyl]thio}-3-ethyl-5,6,7,8-tetrahydro[1]benzothieno[2,3-d]pyrimidin-4(3H)-one (BPOET) stimulates resuscitation. Critically, by screening 4,267 E. coli proteins, we determined that BPOET activates hibernating ribosomes via 23S rRNA pseudouridine synthase RluD, which increases ribosome activity. Corroborating the increased waking with RluD, production of RluD increased the number of active ribosomes in persister cells. Also, inactivating the small RNA RybB which represses rluD led to faster persister resuscitation. Hence, persister cells resuscitate via activation of RluD.

scholarly journals The Cs sec mutants of Escherichia coli reflect the cold sensitivity of protein export itself.

Genetics ◽  
1993 ◽  
Vol 133 (4) ◽  
pp. 763-773 ◽  
Author(s):  
K J Pogliano ◽  
J Beckwith
Keyword(s):  
Escherichia Coli ◽  
Signal Sequence ◽  
Thermal Sensitivity ◽  
Specific Interaction ◽  
Protein Export ◽  
Cold Sensitivity ◽  
Lethal Mutations ◽  
Cold Sensitive ◽  
Sec Proteins ◽  
Striking Effect

Abstract We have found that temperature can have a striking effect upon protein export in Escherichia coli, suggesting that there is a cold-sensitive step in the protein export pathway. Cs mutations comprise the largest class of mutations affecting the membrane-localized Sec proteins SecD, SecE, SecF and SecY. Although some of these mutations could encode cold-labile proteins, this is unlikely to account for the Cs phenotype of most export mutants, as mutations which simply produce lower amounts of SecE protein have the same phenotype. Certain signal sequence mutations affecting maltose binding protein are also cold sensitive for export. These effects appear to arise by a specific interaction of cold with certain export defects. We believe that the Cs sec mutations are representative of a large class of conditional lethal mutations, whose conditional phenotype reflects an underlying thermal sensitivity of the process in which they are involved.

Suppressors of a Cold-Sensitive Mutation in Yeast U4 RNA Define Five Domains in the Splicing Factor Prp8 That Influence Spliceosome Activation

Genetics ◽  
2000 ◽  
Vol 155 (4) ◽  
pp. 1667-1682 ◽  
Author(s):  
Andreas N Kuhn ◽  
David A Brow
Keyword(s):  
Large Scale ◽  
Splicing Factor ◽  
Cold Sensitivity ◽  
Splicing Factors ◽  
Loss Of Function ◽  
U1 Snrnp ◽  
Snrnp Protein ◽  
Cold Sensitive ◽  
Spliceosome Activation ◽  
U4 Rna

AbstractThe highly conserved splicing factor Prp8 has been implicated in multiple stages of the splicing reaction. However, assignment of a specific function to any part of the 280-kD U5 snRNP protein has been difficult, in part because Prp8 lacks recognizable functional or structural motifs. We have used a large-scale screen for Saccharomyces cerevisiae PRP8 alleles that suppress the cold sensitivity caused by U4-cs1, a mutant U4 RNA that blocks U4/U6 unwinding, to identify with high resolution five distinct regions of PRP8 involved in the control of spliceosome activation. Genetic interactions between two of these regions reveal a potential long-range intramolecular fold. Identification of a yeast two-hybrid interaction, together with previously reported results, implicates two other regions in direct and indirect contacts to the U1 snRNP. In contrast to the suppressor mutations in PRP8, loss-of-function mutations in the genes for two other splicing factors implicated in U4/U6 unwinding, Prp44 (Brr2/Rss1/Slt22/Snu246) and Prp24, show synthetic enhancement with U4-cs1. On the basis of these results we propose a model in which allosteric changes in Prp8 initiate spliceosome activation by (1) disrupting contacts between the U1 snRNP and the U4/U6-U5 tri-snRNP and (2) orchestrating the activities of Prp44 and Prp24.

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