RESUMO
LepA is a paralog of EF-G found in all bacteria. Deletion of lepA confers no obvious growth defect in Escherichia coli, and the physiological role of LepA remains unknown. Here, we identify nine strains (ΔdksA, ΔmolR1, ΔrsgA, ΔtatB, ΔtonB, ΔtolR, ΔubiF, ΔubiG or ΔubiH) in which ΔlepA confers a synthetic growth phenotype. These strains are compromised for gene regulation, ribosome assembly, transport and/or respiration, indicating that LepA contributes to these functions in some way. We also use ribosome profiling to deduce the effects of LepA on translation. We find that loss of LepA alters the average ribosome density (ARD) for hundreds of mRNA coding regions in the cell, substantially reducing ARD in many cases. By contrast, only subtle and codon-specific changes in ribosome distribution along mRNA are seen. These data suggest that LepA contributes mainly to the initiation phase of translation. Consistent with this interpretation, the effect of LepA on ARD is related to the sequence of the Shine-Dalgarno region. Global perturbation of gene expression in the ΔlepA mutant likely explains most of its phenotypes.
Assuntos
Proteínas de Escherichia coli/fisiologia , Escherichia coli/genética , Iniciação Traducional da Cadeia Peptídica , Fatores de Iniciação de Peptídeos/fisiologia , Fatores de Iniciação em Procariotos/fisiologia , Domínio Catalítico , Escherichia coli/enzimologia , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , GTP Fosfo-Hidrolases/metabolismo , Deleção de Genes , Elongação Traducional da Cadeia Peptídica , Fatores de Iniciação de Peptídeos/química , Fatores de Iniciação de Peptídeos/genética , Fatores de Iniciação de Peptídeos/metabolismo , Fenótipo , Fatores de Iniciação em Procariotos/química , Fatores de Iniciação em Procariotos/genética , Fatores de Iniciação em Procariotos/metabolismo , Estrutura Terciária de Proteína , RNA Mensageiro/análise , Ribossomos/metabolismoRESUMO
The Fis protein is a nucleoid associated protein that has previously been reported to act negatively in initiation of replication in Escherichia coli. In this work we have examined the influence of this protein on the initiation of replication under different growth conditions using flow cytometry. The Fis protein was found to be increasingly important with increasing growth rate. During multi-fork replication severe under-initiation occurred in cells lacking the Fis protein; the cells initiated at an elevated mass, had fewer origins per cell and the origins were not initiated in synchrony. These results suggest a positive role for the Fis protein in the initiation of replication.
Assuntos
Replicação do DNA/genética , Proteínas de Escherichia coli/fisiologia , Escherichia coli/genética , Fator Proteico para Inversão de Estimulação/fisiologia , Cromossomos Bacterianos/genética , Cromossomos Bacterianos/metabolismo , DNA Bacteriano/genética , Escherichia coli/crescimento & desenvolvimento , Organismos Geneticamente Modificados , Fatores de Iniciação em Procariotos/fisiologia , Iniciação da Transcrição GenéticaRESUMO
RNA polymerase (RNAP) pause sites have been identified in several prokaryotic genes. Although the presumed biological function of RNAP pausing is to allow synchronization of RNAP position with regulatory factor binding and/or RNA folding, a direct causal link between pausing and changes in gene expression has been difficult to establish. RNAP pauses at two sites in the Bacillus subtilis trpEDCFBA operon leader. Pausing at U107 and U144 participates in transcription attenuation and trpE translation control mechanisms, respectively. Substitution of U144 caused a substantial pausing defect in vitro and in vivo. These mutations led to increased trp operon expression that was suppressed by overproduction of TRAP, indicating that pausing at U144 provides additional time for TRAP to bind to the nascent transcript and promote formation of an RNA structure that blocks translation of trpE. These results establish that pausing is capable of playing a role in regulating translation in bacteria.
Assuntos
Bacillus subtilis/genética , Proteínas de Bactérias/metabolismo , RNA Polimerases Dirigidas por DNA/fisiologia , Fatores de Iniciação em Procariotos/genética , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/metabolismo , Fatores de Transcrição/metabolismo , Transcrição Gênica/fisiologia , Sítios de Ligação , Células Cultivadas , Regulação Bacteriana da Expressão Gênica , Modelos Biológicos , Óperon/genética , Fatores de Iniciação em Procariotos/fisiologia , Ligação Proteica , RNA Mensageiro/genética , Fatores de Tempo , Triptofano/metabolismoRESUMO
During initiation of bacterial protein synthesis, messenger RNA and fMet-tRNAfMet bind to the 30S ribosomal subunit together with initiation factors IF1, IF2, and IF3. Docking of the 30S preinitiation complex to the 50S ribosomal subunit results in a peptidyl-transfer competent 70S ribosome. Initiation with an elongator tRNA may lead to frameshift and an aberrant N-terminal sequence in the nascent protein. We show how the occurrence of initiation errors is minimized by (1) recognition of the formyl group by the synergistic action of IF2 and IF1, (2) uniform destabilization of the binding of all tRNAs to the 30S subunit by IF3, and (3) an optimal distance between the Shine-Dalgarno sequence and the initiator codon. We suggest why IF1 is essential for E. coli, discuss the role of the G-C base pairs in the anticodon stem of some tRNAs, and clarify gene expression changes with varying IF3 concentration in the living cell.