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C Squires

Publications and source records attributed to C Squires.

At least 19 recordsLinked to original sources
PubMed1995

rRNA operon multiplicity in Escherichia coli and the physiological implications of rrn inactivation.

Here we present evidence that only five of the seven rRNA operons present in Escherichia coli are necessary to support near-optimal growth on complex media. Seven rrn operons are necessary, however, for rapid adaptation to nutrient and temperature changes, suggesting it is the ability to adapt quickly to changing environmental conditions that has provided the selective pressure for the persistence of seven rrn operons in E. coli. We have also found that one consequence of rrn operon inactivation is a miscoordination of the concentrations of initiation factor IF3 and ribosomes.

Adaptation, Physiological↗
PubMed1993

Depletion of functional ribosomal RNA operons in Escherichia coli causes increased expression of the remaining intact copies.

The synthesis of ribosomal RNA is a complex and highly regulated process. To study this process, we have used deletion-insertions to disrupt sequentially from one to four of the seven rRNA (rrn) operons on the Escherichia coli genome. Inactivation of four rrn operons caused a 2.3-fold increase in the expression of a chloramphenicol acetyl transferase reporter gene fused to the tandem promoters of rrnA and a similar increase in the expression of the trp tRNA gene at the end of rrnC. This reflected enhanced expression of the remaining operons to compensate for having only three intact copies. The elevated expression was caused by an increase in both transcription initiation and RNA polymerase elongation rates specifically on rrn operons and occurred in the absence of changes in the intracellular concentration of ppGpp, suggesting that ppGpp is not involved in the regulation of this phenomenon. We discuss these results in relation to the ribosome feedback inhibition model described by Nomura and coworkers.

Base Sequence↗
PubMed1992

Comparison of the expression of the seven ribosomal RNA operons in Escherichia coli.

We have compared the expression of the seven ribosomal RNA operons (rrn) of Escherichia coli and their responses to a variety of physiological and genetic perturbations. We used a set of rrn promoter fusion constructs in their native chromosomal positions to examine effects of chromosomal location on rrn operon expression and the same set of fusions on lambda lysogens to assay intrinsic promoter strengths independent of chromosome context. In its native chromosomal location, expression of the rrnH operon was significantly lower than expected. This effect was not attributable to weak promoter activity and was dependent on the growth medium. The rrnE operon had reduced promoter activity relative to the other ribosomal operons in minimal medium and thus appears to have abnormal growth rate regulation. The ribosomal RNA operons showed varied responses to amino acid starvation; expression of rrnD was inhibited most. There was only a slight increase in rrn transcription in response to a temperature shift (30 degrees C to 42 degrees C) and the differences between individual operons was very small. The rrnG operon showed a significantly lower response than the other ribosomal RNA operons to a depletion of the rrn transcription activator, Fis, and thus appears to have decreased Fis-mediated transactivation. Finally, the chromosomal fusion strains were used to study the effect on growth rate of inactivating each rrn operon. In fast growth conditions, loss of certain rrn operons caused subtle decreases in growth rate on complex medium.

Base Sequence↗
PubMed1991

Transcriptional termination sequence at the end of the Escherichia coli ribosomal RNA G operon: complex terminators and antitermination.

We have examined the termination region sequence of the rrnG operon and have observed its properties in vivo using a fusion plasmid test system. Transcription of rrnG terminator fragments was also studied in vitro. We found that termination of rrnG transcription is a complex process controlled by a tandem Rho-independent and Rho-dependent terminator arrangement which we designate rrnG-tt'. Together, these two elements were 98% efficient at terminating transcription initiated at the rrnG-P2 promoter. When the two elements were separated, however, we found that the Rho-independent structure was only 59% efficient while the Rho-dependent fragment alone could account for total transcriptional termination of the tandem arrangement. The rrnG termination region was resistant to rrn antitermination and, therefore, possesses some means of stopping antiterminated transcription. The distal rrnG sequence contains several additional noteworthy features; the rrnGt' fragment contains a REP (repetitive extragenic palindromic) sequence and homology with a small unidentified reading frame following rrnE. This sequence is followed by witA, which is homologous to a citrate transport gene, citB. Finally, our sequence, obtained from plasmid pLC23-30, contains a Tn1000 insertion that is absent from the E. coli chromosome. This insertion lies 975 bp beyond the 5S gene and is not involved in the termination events examined in this study.

Amino Acid Sequence↗
PubMed1991

ClpB is the Escherichia coli heat shock protein F84.1.

ClpB is thought to be involved in proteolysis because of its sequence similarity to the ClpA subunit of the ClpA-ClpP protease. It has recently been shown that ClpP is a heat shock protein. Here we show that ClpB is the Escherichia coli heat shock protein F84.1. The F84.1 protein was overproduced in strains containing the clpB gene on a plasmid and was absent from two-dimensional gels from a clpB null mutation. Besides possessing a slower growth rate at 44 degrees C, the null mutant strain had a higher rate of death at 50 degrees C. We used reverse transcription of in vivo mRNA to show that the clpB gene was expressed from a sigma 32-specific promoter consensus sequence at both 37 and 42 degrees C. We noted that the clpB+ gene also caused the appearance of a second protein spot, F68.5, on two-dimensional gels. This spot was approximately 147 amino acids smaller than F84.1 and most probably is the result of a second translational start on the clpB mRNA. F68.5 can be observed on many published two-dimensional gels of heat-induced E. coli proteins, but the original catalog of 17 heat shock proteins did not include this spot.

Base Sequence↗
PubMed1990

Antitermination of characterized transcriptional terminators by the Escherichia coli rrnG leader region.

We have used a plasmid antitermination test system to examine the response of an Escherichia coli rRNA operon antiterminator to transcription through Rho-dependent and Rho-independent terminator-containing fragments. We also monitored transcription through multiple copies of a terminator to explore the mechanism of rrn antitermination. Four principal observations were made about antitermination and transcriptional terminators. (1) The rrn antiterminator mediated efficient transcription through Rho-dependent terminators. (2) Under the influence of the rrn antiterminator, RNA polymerase transcribed through two and three copies of the Rho-dependent 16 S----terminator with nearly the same efficiency as through one. (3) The antiterminator had less effect on fragments containing Rho-independent terminators; the rpoC t fragment and three fragments derived from the rrnB terminator region stopped antiterminated transcription. Four other Rho-independent terminator fragments were weakly antiterminated in our test system. (4) Surprisingly, the strength of these terminator fragments was not strongly related to properties such as the -delta G or number of trailing uridine residues of their canonical Rho-independent structures, but appears to be related to additional downstream terminators. We have drawn the following conclusions from these experiments. First, that ribosomal antitermination primarily reverses Rho-dependent termination by modifying the RNA polymerase elongation complex. Transcription through a 1700 nucleotide, multiple terminator sequence showed that the antiterminator caused persistent changes in the transcription process. Second, that fragments derived from the Rho-independent rrnB and rpoBC terminator regions can effectively stop antiterminated transcription. Third, that efficient in vivo termination may often involve regions with complex multiple terminators.

Cloning, Molecular↗
PubMed1990

Conservation of the regulatory subunit for the Clp ATP-dependent protease in prokaryotes and eukaryotes.

Bacteria, tomatoes, and trypanosomes all contain genes for a large protein with extensive homology to the regulatory subunit, ClpA, of the ATP-dependent protease of Escherichia coli, Clp. All members of the family have between 756 and 926 amino acids and contain two large regions, of 233 and 192 amino acids, each containing consensus sequences for nucleotide binding. Within these regions there is at least 85% similarity between the most distant members of the family. The high degree of similarity among the ClpA-like proteins suggests that Clp-like proteases are likely to be important participants in energy-dependent proteolysis in prokaryotic and eukaryotic cells.

ATP-Dependent Proteases↗
PubMed1989

Ribosomal RNA operon anti-termination. Function of leader and spacer region box B-box A sequences and their conservation in diverse micro-organisms.

All Escherichia coli rrn operons show a common motif in which anti-terminator box B-box A sequences occur twice, first in the leader and again in the 16 S-23 S spacer. In this study we have analyzed several aspects of rrn anti-termination by leader and spacer anti-terminator sequences. Using DNA synthesis and a plasmid test system, we incorporated random changes into the leader anti-terminator region and examined these mutations for their ability to read through a strong terminator. We also examined anti-termination by synthetic box A and by rrn spacer region sequences. Information derived from these experiments was used to search the rrn sequences of other micro-organisms for possible anti-termination features. Our principal conclusions were that: (1) box A was sufficient for terminator readthrough; (2) we could show no positive requirement for box B in our test system; (3) many of the negative anti-terminator mutations caused a promoter up-effect in the absence of a terminator; (4) the search of rrn operons from other micro-organisms revealed that anti-terminator-like box B-box A sequences exist in leader and spacer regions of both eubacteria and archaebacteria. The frequent occurrence of this pattern suggested that the E. coli rrn anti-termination motif is widespread in nature and has been conserved in microbial evolution.

Base Sequence↗
PubMed1988

Loss of the spacer loop sequence from the rrnB operon in the Escherichia coli K-12 subline that bears the relA1 mutation.

A polymorphism affecting the spacer region of the rrnB rRNA operon is described. Strains from a major Escherichia coli K-12 subbranch are missing a 106-nucleotide portion of the rrnB 16S-to-23S spacer, and a 20-nucleotide sequence is found in its place. We have called this mutant operon rrnB2. The rrnB2 spacer was most probably derived from either rrnC or rrnE. This alteration of rrnB may have occurred by a recombinational exchange or by gene conversion. In the genealogy of E. coli K-12 strains, the appearance of rrnB2 is associated with the spontaneous occurrence of the first relaxed mutation, but attempts to show a selective relationship between the two mutational events have had negative results. The sequences of the rrnG and rrnC 16S-to-23S spacers have also been determined and their comparisons to the other rrn operons encoding tRNAGlu2 are presented.

Base Sequence↗
PubMed1987

In vivo translation of a region within the rrnB 16S rRNA gene of Escherichia coli.

In this study we show that a segment of the Escherichia coli rrnB 16S gene can be translated in vivo. Other laboratories have previously reported that there are internal transcription and translation signals and open reading frames within the E. coli rrnB rRNA operon. Their studies revealed a translation start signal followed by a 252-base-pair open reading frame (ORF16) within the 16S gene and detected a promoter (p16) in the same general region by using in vitro RNA polymerase binding and transcription initiation assays. By using plasmid gene fusions of ORF16 to lacZ we showed that an ORF16'-'beta-galactosidase fusion protein was made in vivo. Transcripts encoding the fusion protein were expressed either from the rrnB p1p2 control region or from a hybrid trp-lac promoter (tacP), but the amount of expression was considerably less than for a lacZ control plasmid. We used fusions to the cat gene to show that p16 is one-half as active as lacP. Deletions were used to show that p16 is located within ORF16 and thus cannot promote a transcript encoding the ORF16 peptide. A comparison of sequences from different organisms shows that ORF16 and p16 lie in a highly conserved region of the procaryotic 16S RNA structure. The first 20 amino acids of ORF16 are conserved in most eubacterial and plant organellar sequences, and promoter activity has been detected in this region of the Caulobacter crescentus sequence by other workers.

Codon↗
PubMed1984

Antitermination of E. coli rRNA transcription is caused by a control region segment containing lambda nut-like sequences.

We have localized the antitermination system involved in E. coli ribosomal RNA transcription and compared it with antitermination in the lamboid bacteriophages. In vivo experiments with gene-fusion plasmids were used to examine the ability of specific areas of the rrnG control region to convert an ordinary transcription complex into antitermination transcription complex. A 67 bp restriction fragment immediately following the rrnG P2 promoter decreased transcription termination about 50%. This fragment contains box A-, box B-, and box C-like sequences similar to those in lambda nut loci. It also caused transcripts from lac and hybrid trp-lac promoters to read through a transcription terminator. Translation through the 67 bp segment or reversal of its orientation resulted in complete loss of antitermination activity. We conclude that the E. coli ribosomal RNA operons possess an antitermination system similar to that used by the bacteriophage lambda.

Bacteriophage lambda↗
PubMed1984

Translational coupling of the trpB and trpA genes in the Escherichia coli tryptophan operon.

We investigated whether there is translational coupling between the tryptophan operon trpB and trpA genes in Escherichia coli. A trp-lac fusion system was used in which part of the trpA gene is fused to the lacZ gene. This fusion protein has the translation initiation site of trpA but retains beta-galactosidase activity. We introduced a frameshift mutation early in trpB and measured its effect on transcription and translation of the trp-lac fusion. The mutation resulted in a 10-fold drop in beta-galactosidase activity but only a 2-fold drop in lacZ mRNA or galactoside transacetylase levels. An rho mutation restored the lacZ mRNA and transacetylase levels to those of the control but only increased the beta-galactosidase level to 20% that of the control. We conclude from these results that if the trpB gene is not translated, efficient translation of the trpA'-lac'Z mRNA does not occur and, thus, that these genes are translationally coupled. The implication of this finding for other studies with gene fusions is discussed.

Acetyltransferases↗
PubMed1984

Evidence for antitermination in Escherichia coli RRNA transcription.

The stable RNA operons of Escherichia coli do not exhibit polarity, even though they make an RNA product that is not translated. By contrast, most E. coli operons that specify proteins exhibit polarity if their translation is interrupted. The transcriptional component of this polarity depends on the action of Rho protein on the exposed mRNA, which results in premature transcription termination. Here we examine how a stable RNA operon (rrnG) transcript is protected from the Rho protein-mediated polarity response. We compared transcription from the ara and the rrnG promoters through a 16S DNA segment. In each case, the promoter-16S sequences were joined to a trp-lac fusion, and lacZ mRNA was examined in rho+ and rho-115 strains. We found significant Rho protein-dependent termination of transcripts from the ara promoter but little or no Rho protein effect on transcription from the rrnG promoter. We concluded that the transcript of the 16S ribosomal DNA segment does contain Rho protein-dependent transcription terminators, but there is an antitermination system in the rrnG control region that allows it to transcribe through those terminators.

Base Sequence↗
PubMed1982

The regulatory region of the divergent argECBH operon in Escherichia coli K-12.

The nucleotide sequence of the control region of the divergent argECBH operon has been established in the wild type and in mutants affecting expression of these genes. The argE and argCBH promoters face each other and overlap with an operator region containing two domains which may act as distinct repressor binding sites. A long leader sequence - not involved in attenuation - precedes argCBH. Overlapping of the argCBH promoter and the region involved in ribosome mobilization for argE translation explains the dual effect of some mutations. Mutations causing semi-constitutive expression of argE improve putative promoter sequences within argC. Implications of these results regarding control mechanisms in amino acid biosynthesis and their evolution are discussed.

Base Sequence↗
PubMed1982

Nucleotide sequence of the rrnG ribosomal RNA promoter region of Escherichia coli.

The primary structure of the promoter region for a ribosomal RNA transcription unit (rrnG) of Escherichia coli K12 has been determined. The sequence was obtained from 1 1.5 kbp EcoRI fragment derived from the hybrid plasmid pLC23-30. This fragment contains 455 bp preceding P1 of the rrnG promoter region and 674 bp of the rrnG 16S RNA gene. The sequence before the rrnG promoter region contains an open reading frame (ORF-BG) followed by a possible hairpin structure that resembles other known transcription terminators. The sequence of the rrnG promoter region is similar but not identical to that of rrnA and rrnB. Several minor differences between the sequences of the 16S RNA genes of rrnG and rrnB were also noted. In addition, sequences were found that could generate special structures involving the promoter regions of rrn loci. Such structures are described and their possible involvement in the regulation of ribosomal RNA synthesis is discussed.

Base Sequence↗
PubMed1981

Nucleotide sequence at the end of the gene for the RNA polymerase beta' subunit (rpoC).

We have determined the DNA sequence surrounding the transcription terminator following rpoC, the gene that codes for the beta' subunit of RNA polymerase in E. coli K12. The 2044 bp sequence obtained contains the distal 335 codons of rpoC followed by a 212 bp non-coding region and a second open reading frame (ORFa) of 179 codons. The final 181 nucleotides of the sequence form the 5' end of a third open reading frame (ORFb). The in vivo 3' end of the rpoC mRNA was located by analysis of RNA/DNA hybrids cleaved with nuclease S1 (S1 mapping). These results indicated that the major transcription termination of the rplJL-rpoBC transcription unit occurs a short distance past the translation stop codon for rpoC. Four regions of symmetry, suggesting secondary structure in the mRNA, were found in the DNA sequence near the rpoC translation termination codon. The last of these hairpin structures is similar to other rho-independent transcription terminators and its 3' end coincides with the end of the rpoC mRNA as predicted by S1-mapping. Inspection of the open reading frames indicates that rpoC uses a high percentage of codons that are recognized by the major tRNA species of E. coli while ORFa and ORFb contain many codons recognized by minor tRNA species. ORFa specifies a very basic peptide.

Base Sequence↗