Genetic and physical analysis of the thioredoxin (trxA) gene of Escherichia coli K-12

We present evidence that the expression of rho is regulated by rho-dependent attenuation of transcription. Gene fusion analysis with nested series of deletions of rho indicated that the transcription of rho is attenuated in a rho-dependent manner in the leader region and that neither a read-through transcription from the upstream gene, trxA, nor a modulation of transcription initiation of the rho promoter is involved in the self-control of rho. Si mapping and Northern hybridization analyses localized at least six transcription attenuation or termination sites in the region ranging from the 3' end of the trxA structural gene to the middle of the rho structural gene. Among them, the most upstream site overlapping the rho promoter sequence was assigned to the terminator for the trxA gene, and the second and third sites, mapping about 80 and 50 nucleotides upstream from the start codon of rho, were suggested to function as the major attenuation sites for regulation of the rho expression. Further, the start points of the trxA and rho RNAs were determined in an in vitro transcription system to be located 111 nucleotides (U) and 255 nucleotides (G) Kushner, Gene 32:399408, 1984).The termination of transcription plays profound roles in the regulation of gene expression in procaryotes. Termination of RNA synthesis generally occurs at the ends of operons so as to ensure independent expression of neighboring genes. In addition, it can also occur within operons to allow differential expression of genes within an operon. In Escherichia coli, several protein factors which participate in transcription termination or antitermination have been identified: Rho, NusA, NusB, and NusE (14,21,22,25,26,30,47,56).Rho protein causes termination of transcription catalyzed by E. coli RNA polymerase at certain specific sites on DNA template (47). These sites are called Rho-dependent terminators, as opposed to Rho-independent terminators which are recognized by RNA polymerase without the aid of Rho. Several Rho-dependent terminators have been found at different positions in various operons. For example, trp-t' (57) and the tyrT terminator (33) are located at the ends of their operons, while tLl, tRl, and tR2 are within the leftward and rightward operons of bacteriophage X, respectively (47). It has also been shown that mutational polarity involves Rho-dependent terminators residitng within a structural gene.Such terminators are normally nonfunctional, but are activated when preceding translation is arrested at a nonsense codon introduced by mutation (1, 13). A number of rho mutants of E. coli have been isolated that are defective in Rho-dependent transcription termination (12,28,29,31,46). Some of them are conditional-lethal, suggesting that Rho is essential for cell growth. (27, 45). Conversely, decreased Rho levels have been observed with a new class of rho mutants which is hyperactive in termination (53). These observations suggest that the expression of rho is autogenously regulated. In view of the role of Rho as a transcription term...

mentioning

confidence: 99%

Autogenous regulation of the gene for transcription termination factor rho in Escherichia coli: localization and function of its attenuators

Matsumoto¹,

Shigesada²,

Hirano³

et al. 1986

“…The two-to threefold decrease in percent trxA mRNA is consistent with the decrease in total RNA per cell (Table 1) and contrasts to the increase in percent cat mRNA. These results would suggest that cyclic AMP concentrations are not elevated in the slowly growing cells as compared to those in cells with the 97-min generation time, assuming that trxA is regulated by catabolite repression (31,44). The high level of cat mRNA present in slowly growing cells would not be due, therefore, to either changes in catabolite repression or generalized aberrant regulation of transcription.…”

Section: Methodsmentioning

confidence: 90%

“…trxA is thought to be under catabolite repression (31,44) like cat (15,27) and provides a convenient control for monitoring the potential effects of changes in this regulation. When the same RNA samples that were used for cat quantitation were used for trxA, the level of trxA mRNA decreased from approximately 0.06 to 0.02% as the growth rate decreased (Table 3).…”

Section: Methodsmentioning

confidence: 99%

A novel transcriptional response by the cat gene during slow growth of Escherichia coli

Meyer

Schottel

1991

A novel response to growth rate was found with expression of the chloramphenicol acetyltransferase (cat) gene in Escherichia coli. The amount of cat mRNA relative to total RNA increased about 11-fold as growth rates decreased 5-to 6-fold, without an increase in translation. The accumulation of cat mRNA was in contrast to decreased cellular concentrations of total RNA, trxA, ompA, or 23S rRNA as the growth rate decreased and was not due to changes in gene dosage or mRNA stability. Stability of the cat mRNA does not appear to be regulated by growth rate. No significant change in either chemical or functional stability was observed within a five-to sixfold range of growth rates when chemostat-grown cells were used. However, cat mRNA stability was affected by growth medium composition. The half-life of cat mRNA decreased about threefold, with an approximate fourfold increase in generation time due to changes in growth medium. Transcriptional studies have indicated that accumulation of cat mRNA at slow growth rates is the result of a specific transcriptional response to changes in cellular generation times. We propose that increases in the cellular concentration of a specific message at slow growth rates may reflect an additional type of survival response in E. coli.Regulation of gene expression in Escherichia coli frequently involves an interplay of transcriptional and posttranscriptional events that can be influenced by several parameters of growth. Growth rate is one of the parameters that has been implicated as an important factor affecting gene transcription and the stability of particular mRNAs in E. coli. At the transcriptional level, growth rate has been shown to affect tRNA synthesis (17) and to be involved in the proposed negative feedback regulation of rRNA synthesis (22) where transcription decreases with decreasing growth rate. Changes in transcription of a particular gene can obviously impact gene expression.Even though growth rate does not apparently influence the stability of most transcripts (5,12,20,21,37), it has been proposed to regulate the stability of the ompA and chloramphenicol acetyltransferase (cat) mRNAs in E. coli (32,35). Changes in message stability can affect the levels of protein synthesized from that transcript as seen for ompA (18), the puf operon of Rhodobacter capsulatus (11), and the pap (2) and ars (36) operons of E. coli. Therefore, in addition to transcriptional regulation, changes in mRNA stability due to changes in growth rate could have a critical effect on synthesis of the corresponding protein. Additional evidence of growth rate effects on gene expression can be seen in the increasing or decreasing levels of protein synthesis of numerous but largely uncharacterized E. coli proteins (38).Our interest has been in the events that regulate the stability of mRNA and thus expression of the cat gene. Although the stability of cat mRNA is reported to decrease with decreasing growth rate, the conditions used to vary growth rate involved carbon source changes (35). Since variations...

“…Therefore, it is probable that readthrough transcription from the tetR gene is sufficient for Rho protein synthesis. The trxA transcript has been shown to overlap the -35 region of rho (23,34,37,43). On the basis of the ability of AMC501 to support the growth of phage T7, insertion of TnJO into the 3' noncoding region of the trxA transcript does not have a detrimental effect upon thioredoxin synthesis.…”

Section: Resultsmentioning

confidence: 99%

A novel rho promoter::Tn10 mutation suppresses and ftsQ1(Ts) missense mutation in an essential Escherichia coli cell division gene by a mechanism not involving polarity suppression

Storts

Markovitz

1991

An extragenic suppressor of the Escherichia coli cell division gene ftsQl(Ts) was isolated. The suppressor is a TnWO insertion into the -35 promoter consensus sequence of the rho gene, designated rho promoter:: TnlO. TheftsQl(Ts) mutation was also suppressed by the rho4 mutant allele. The rho promoter::TnlO strain does not exhibit rho mutant polarity suppressor phenotypes. In addition, overexpression of the ftsQl(Ts) mutation does not reverse temperature sensitivity. Furthermore, DNA sequence analysis of theftsQl(Ts) allele revealed that the salt-remediable, temperature-sensitive phenotype arose from a single missense mutation. The most striking phenotype of the rho promoter::TnlO mutant strain is an increase in the level of negative supercoiling. On the basis of these observations, we conclude that the ftsQl(Ts) mutation may be suppressed by a change in supercoiling.A large number of genes have been implicated in Escherichia coli cell division. Most of the characterized genes are clustered around the 2-and 76-min regions of the E. coli chromosome (3; for reviews, see references 8 and 9). The 2-min region has been extensively studied and includes the ftsQ, ftsA, ftsZ (sulB, sfiB), and ftsI (sep, pbpB) genes. This region has been sequenced (33,35,36,47,48), and the individual protein products have been characterized (8,9,39,48). With the exception of the ftsI gene product (8, 9), little is known about the function of the individual components. Ward and Lutkenhaus (44) demonstrated that overexpression offtsZ results in minicell formation. This observation and the demonstration that FtsZ is the target of the cell division inhibitor SulA (8,9) have led to the conclusion that FtsZ may be a rate-limiting component in cell division.TheftsQ gene product is an essential component of E. coli cell division. Conditional lethal mutations have been isolated that filament and die at restrictive temperatures on medium without salt (4). We recently demonstrated that theftsQ gene encodes a stable, inner membrane-associated protein of 31,400 Da that is expressed at low levels in the cell (39). However, overproduction of the FtsQ protein had no apparent affect upon cellular morphology in wild-type cells such as strain AMC290 (Table 1).To further investigate the role of FtsQ in cell division and to examine potential interactions with other cell division proteins, we isolated an extragenic suppressor offtsQl(Ts).In this report, we characterize the ftsQJ(Ts) mutation and demonstrate that mutations in either the rho gene or its promoter are capable of suppressing theftsQJ(Ts) mutation. MATERIALS AND METHODSMedia, bacterial strains, and bacteriophages. The bacterial strains and phages used in this study are listed in Table 1