Cell cycle arrest in Era GTPase mutants: a potential growth rate‐regulated checkpoint in <i>Escherichia coli</i>

Britton, Robert A.; Powell, Bradford S.; Dasgupta, Santanu; Sun, Qin; Margolin, William; Lupski, James R.; Court, Donald L.

doi:10.1046/j.1365-2958.1998.00719.x

Cited by 119 publications

(166 citation statements)

References 45 publications

Supporting

Mentioning

160

Contrasting

Order By: Relevance

“…Remarkable equality between expression of RNase III and Era, the product of the second gene in the three-gene rnc operon, suggest that rnc and era are transcriptionally and translationally coupled Matsunaga et al, 1996). The recent identification of Era as a potential cell cycle regulator (Britton et al, 1997; indicates that RNase III and Era may collaborate to tie the growth rate to the cell cycle through their tightly linked co-expression (Britton et al, 1998).…”

Section: Regulation Of the Rnc Operonmentioning

confidence: 99%

Genetic uncoupling of the dsRNA‐binding and RNA cleavage activities of the Escherichia coli endoribonuclease RNase III — the effect of dsRNA binding on gene expression

Dasgupta

Fernández

Kameyama

et al. 1998

Molecular Microbiology

108

View full text Add to dashboard Cite

SummaryRNase III, a double-stranded RNA-specific endonuclease, is proposed to be one of Escherichia coli 's global regulators because of its ability to affect the expression of a large number of unrelated genes by influencing post-transcriptional control of mRNA stability or mRNA translational efficiency. Here, we describe the phenotypes of bacteria carrying point mutations in rnc, the gene encoding RNase III. The substrate recognition and RNA-processing properties of mutant proteins were analysed in vivo by measuring expression from known RNase III-modulated genes and in vitro from the proteins' binding and cleavage activities on known double-stranded RNA substrates. Our results show that although the point mutation rnc70 exhibited all the usual rnc null-like phenotypes, unlike other mutations, it was dominant over the wild-type allele. Multicopy expression of rnc70 could suppress a lethal phenotype of the wild-type rnc allele in a certain genetic background; it could also inhibit the RNase III-mediated activation of N gene translation by competing for the RNA-binding site of the wild-type endonuclease. The mutant protein failed to cleave the standard RNase III substrates in vitro but exhibited an affinity for double-stranded RNA when passed through poly(rI):poly(rC) columns. Filter binding and gel-shift assays with purified Rnc70 showed that the mutant protein binds to known RNase III mRNA substrates in a site-specific manner. In vitro processing reactions with purified enzyme and labelled RNA showed that the in vivo dominant effect of the mutant enzyme over the wild-type was not necessarily caused by formation of mixed dimers. Thus, the rnc70 mutation generates a mutant RNase III with impaired endonucleolytic activity but without blocking its ability to recognize and bind double-stranded RNA substrates.

show abstract

Section: Regulation Of the Rnc Operonmentioning

confidence: 99%

Genetic uncoupling of the dsRNA‐binding and RNA cleavage activities of the Escherichia coli endoribonuclease RNase III — the effect of dsRNA binding on gene expression

Dasgupta

Fernández

Kameyama

et al. 1998

Molecular Microbiology

108

View full text Add to dashboard Cite

show abstract

“…Later, detailed sequence analysis suggested that Era is not a RAS-like protein (Chen et af., 1990). Recently, a human homologue of Era was reported that is distincr from RAS proteins (Britton et al, 1998 The GenBank accession number for the nucleotide sequence of the Streptococcu:: pneumoniae era gene is AF07281. negative bacteria, and Mycopfasma spp. (Ahnn et al, 1986;Fleischmann et al, 1995;Fraser et al, 1995;Kawabata et al, 1997;Zuber et al, 1990Zuber et al, , 1997, and is essential for bacterial growth (Gollop & March, 1991b;Inada et al, 1989;March et al, 1988;Sat0 et al, 1998;Takiff et al, 1989Takiff et al, , 1992.…”

Section: Introductionmentioning

confidence: 99%

“…However, the biochemical basis for their failure to complement the E. coli era mutants remains unknown as biochemical characterization of these mutant Era proteins was not described Pillutla et a!., 1995). Genetic studies showed that depletion of the cellular amount of Era inhibited the growth of E. coli cells and caused the cells to elongate (Britton et al, 1997(Britton et al, , 1998Gollop & March, 1991b;March et al, 1988). Also, the viability of these cells was drastically reduced upon repression of Era production (Gollop & March, 1991b;Lerner & March, 1991 ;March et al, 1988).…”

Section: Introductionmentioning

confidence: 99%

“…Under these conditions, cell constriction or septum formation was not observed (Gollop & March, 1991b), suggesting that Era may play a role in cell cycle control. More recently, genetic studies also demonstrated that a mutation in the E. coli era gene suppressed temperature-sensitive mutations in the genes encoding chromosome replication and partitioning enzymes such as dnaB, dnaG, gyrB, mukB, parB and parC, but not those of the cell division genes ftsA, ftsZ and ftsZ (Britton et al, 1997(Britton et al, , 1998. Some of the era mutant cells were found to contain two or four segregated nucleoids (Britton et al, 1997(Britton et al, , 1998.…”

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

Biochemical and molecular analyses of the C-terminal domain of Era GTPase from Streptococcus pneumoniae

Zhao¹,

Meier²,

Peery³

et al. 1999

Microbiology

View full text Add to dashboard Cite

Era, an essential GTPase, is present in many bacteria and Mycoplasma spp. and appears to play a major role in the cell cycle and other cellular processes. To further understand its function, an era gene from Streptococcus pneumoniae was identified and cloned, and a mutant era gene with a deletion of 68 codons from its 3'4erminus was constructed. The truncated Era protein was then purified and characterized, and the ability of the truncated era gene to complement an Escherichia coli mutant strain defective in Era production was examined. Like the full-length Era protein, the truncated Era protein was able to bind and hydrolyse GTP, but its binding activity was increased twofold and its hydrolytic activity was reduced sevenfold when compared with those of the full-length Era protein. Unlike the full-length Era protein, the truncated Era protein lost its ability to bind to the E. coli cytoplasmic membrane. The fulllength era gene was able to complement the €. coli mutant deficient in Era production when carried on pACYCl84, while the truncated era gene failed to do so when carried on pACYCl84, pBR322 or pUC18. The cellular amounts of the truncated Era and the full-length Era proteins in €. coli and S. pneumoniae, respectively, were then determined by Western blot analysis. In addition, the minimal amount of the S. pneumoniae Era protein needed for complementation of the E. coli mutant was also measured. Taken together, these results suggest that the C-terminus of the Era protein might be responsible for the binding of the protein to the cytoplasmic membrane and be essential for function.

show abstract

“…The elongated cells contained two or four segregated nucleoids [3,4]. In addition, a mutant allele of E. coli era suppressed several temperaturesensitive alleles of the genes affecting chromosome replication and partitioning [3,4]. Thus, Era plays a role in DNA replication and cell cycle progression.…”

mentioning

confidence: 99%

Analysis of the interaction of 16S rRNA and cytoplasmic membrane with the C-terminal part of the Streptococcus pneumoniae Era GTPase

Hang¹,

Meier²,

Zhao³

2001

Eur J Biochem

View full text Add to dashboard Cite

Era, an essential GTPase, plays a regulatory role in several cellular processes. The Era protein of Streptococcus pneumoniae has recently been shown to bind to 16S rRNA and the cytoplasmic membrane. However, exact locations of Era responsible for RNA-and membrane-binding were unknown. To identify the regions in Era that interact with the RNA and membrane, the C-terminal part of S. pneumoniae Era was systematically deleted while the N-terminal part, responsible for the GTPase activity of the protein, was kept intact. The resulting truncated Era proteins were purified and characterized. The C-terminal deletion of 9 or 19 amino-acid residues did not affect 16S rRNA-binding activity while further deletions of the C-terminus (29-114 amino-acid residues) abolished the activity. These results indicate that the integrity of the putative KH domain of Era, spanning the amino-acid residues between < 22-83 from the C-terminus, is required for 16S rRNA-binding. Furthermore, the Era proteins with a deletion up to 45 residues from the C-terminus retained membrane-binding activity, but longer deletions significantly reduced the activity. These results indicate that part of the putative KH domain is also required for membrane-binding. Thus, these results indicate for the first time that the regions critical for the membrane-and 16S rRNA-binding activities of Era overlap. The era gene with a deletion of 9 or 19 codons from its 3 0 terminus complemented an Escherishia coli mutant strain deficient in Era production whereas the genes with longer deletions failed to do so, thereby indicating that the KH domain is essential for Era function. Taken together, the results of this study indicate that the putative KH domain is required for 16S rRNA-binding activity and that part of the KH domain is also required for membrane-binding activity. The results also suggest that the interaction between Era and 16S rRNA is essential for bacterial growth.

show abstract

Cell cycle arrest in Era GTPase mutants: a potential growth rate‐regulated checkpoint in Escherichia coli

Cited by 119 publications

References 45 publications

Genetic uncoupling of the dsRNA‐binding and RNA cleavage activities of the Escherichia coli endoribonuclease RNase III — the effect of dsRNA binding on gene expression

Genetic uncoupling of the dsRNA‐binding and RNA cleavage activities of the Escherichia coli endoribonuclease RNase III — the effect of dsRNA binding on gene expression

Biochemical and molecular analyses of the C-terminal domain of Era GTPase from Streptococcus pneumoniae

Analysis of the interaction of 16S rRNA and cytoplasmic membrane with the C-terminal part of the Streptococcus pneumoniae Era GTPase

Contact Info

Product

Resources

About