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T M Henkin

Publications and source records attributed to T M Henkin.

At least 19 recordsLinked to original sources
PubMed2001

The GA motif: an RNA element common to bacterial antitermination systems, rRNA, and eukaryotic RNAs.

Two different transcription termination control mechanisms, the T box and S box systems, are used to regulate transcription of many bacterial aminoacyl-tRNA synthetase, amino acid biosynthesis, and amino acid transport genes. Both of these regulatory mechanisms involve an untranslated mRNA leader region capable of adopting alternate structural conformations that result in transcription termination or transcription elongation into the downstream region. Comparative analyses revealed a small RNA secondary structural element, designated the GA motif, that is highly conserved in both T box and S box leader sequences. The motif consists of two short helices separated by an asymmetric internal loop, with highly conserved GA dinucleotide sequences on either side of the internal loop. Site-directed mutagenesis of this motif in model T and S box leader sequences indicated that it is essential for transcriptional regulation in both systems. This motif is similar to the binding site of yeast ribosomal protein L30, the Snu13p binding sites found in U4 snRNA and box C/D snoRNAs, and two elements in 23S rRNA.

Bacteria↗
PubMed2001

Transcriptional activation of the Bacillus subtilis ackA promoter requires sequences upstream of the CcpA binding site.

Carbon catabolite protein A (CcpA) is a global regulator of carbon metabolism in gram-positive bacteria, repressing transcription of genes for the utilization of secondary carbon sources in the presence of a readily metabolized carbon source and activating transcription of genes, such as ackA and pta, that are required for carbon excretion. The promoter region of the Bacillus subtilis ackA gene contains two catabolite responsive elements (cre sites), of which only the site closest to the promoter (cre2) binds CcpA to activate transcription. A region immediately upstream of the cre2 site is also important for transcriptional activation. The required elements in this region were further defined by mutagenesis. CcpA binds to the ackA promoter region in gel shift assays even in the presence of mutations in the upstream element that block transcriptional activation, indicating that this region has a function other than promoting binding of CcpA.

Bacillus subtilis↗
PubMed2000

Transcription termination control in bacteria.

Transcription termination is a dynamic process and is subject to control at a number of levels. New information about the molecular mechanisms of transcription elongation and termination, as well as new insights into protein-RNA interactions, are providing a framework for increased understanding of the molecular details of transcription termination control.

Bacteria↗
PubMed2000

tRNA determinants for transcription antitermination of the Bacillus subtilis tyrS gene.

Transcriptional regulation of the T box family of aminoacyl-tRNA synthetase and amino acid biosynthesis genes in Gram-positive bacteria is mediated by a conserved transcription antitermination system, in which readthrough of a termination site in the leader region of the mRNA is directed by a specific interaction with the cognate uncharged tRNA. The specificity of this interaction is determined in part by pairing of the anticodon of the tRNA with a "specifier sequence" in the leader, a codon representing the appropriate amino acid, as well as by pairing of the acceptor end of the tRNA with an unpaired region of the antiterminator. Previous studies have indicated that although these interactions are necessary for antitermination, they are unlikely to be sufficient. In the current study, the effect of multiple mutations in tRNA(Tyr) on readthrough of the tyrS leader region terminator, independent of other tRNA functions, was assessed using a system for in vivo expression of pools of tRNA variants; this system may be generally useful for in vivo expression of RNAs with defined end points. Although alterations in helical regions of tRNA(Tyr) that did not perturb base pairing were generally permitted, substitutions affecting conserved features of tRNAs were not. The long variable arm of tRNA(Tyr) could be replaced by either a short variable arm or a long insertion of a stable stem-loop structure. These results indicate that the tRNA-leader RNA interaction is highly constrained, and is likely to involve recognition of the overall tertiary structure of the tRNA.

Amino Acyl-tRNA Synthetases↗
PubMed2000

Bacillus subtilis ccpA gene mutants specifically defective in activation of acetoin biosynthesis.

A large number of carbon source utilization pathways are repressed in Bacillus subtilis by the global regulator CcpA, which also acts as an activator of carbon excretion pathways during growth in media containing glucose. In this study, CcpA mutants defective in transcriptional activation of the alsSD operon, which is involved in acetoin biosynthesis, were identified. These mutants retained normal glucose repression of amyE, encoding alpha-amylase, and acsA, encoding acetyl-coenzyme A synthetase, and normal activation of ackA, which is involved in acetate excretion; in these ccpA mutants the CcpA functions of activation of the acetate and acetoin excretion pathways appear to be separated.

Acetates↗
PubMed1999

Metabolic imbalance and sporulation in an isocitrate dehydrogenase mutant of Bacillus subtilis.

A Bacillus subtilis mutant with a deletion in the citC gene, encoding isocitrate dehydrogenase, the third enzyme of the tricarboxylic acid branch of the Krebs cycle, exhibited reduced growth yield in broth medium and had greatly reduced ability to sporulate compared to the wild type due to a block at stage I, i.e., a failure to form the polar division septum. In early stationary phase, mutant cells accumulated intracellular and extracellular concentrations of citrate and isocitrate that were at least 15-fold higher than in wild-type cells. The growth and sporulation defects of the mutant could be partially bypassed by deletion of the major citrate synthase gene (citZ), by raising the pH of the medium, or by supplementation of the medium with certain divalent cations, suggesting that abnormal accumulation of citrate affects survival of stationary-phase cells and sporulation by lowering extracellular pH and chelating metal ions. While these genetic and environmental alterations were not sufficient to allow the majority of the mutant cell population to pass the stage I block (lack of asymmetric septum formation), introduction of the sof-1 mutant form of the Spo0A transcription factor, when coupled with a reduction in citrate synthesis, restored sporulation gene expression and spore formation nearly to wild-type levels. Thus, the primary factor inhibiting sporulation in a citC mutant is abnormally high accumulation of citrate, but relief of this metabolic defect is not by itself sufficient to restore competence for sporulation.

Adenosine Triphosphate↗
PubMed1998

The S box regulon: a new global transcription termination control system for methionine and cysteine biosynthesis genes in gram-positive bacteria.

The molecular mechanisms for regulation of the genes involved in the biosynthesis of methionine and cysteine are poorly characterized in Bacillus subtilis. Analyses of the recently completed B. subtilis genome revealed 11 copies of a highly conserved motif. In all cases, this motif was located in the leader region of putative transcriptional units, upstream of coding sequences that included genes involved in methionine or cysteine biosynthesis. Additional copies were identified in Clostridium acetobutylicum and Staphylococcus aureus, indicating conservation in other Gram-positive genera. The motif includes an element resembling an intrinsic transcriptional terminator, suggesting that regulation might be controlled at the level of premature termination of transcription. The 5' portion of all of the leaders could fold into a conserved complex structure. Analysis of the yitJ gene, which is homologous to Escherichia coli metH and metF, revealed that expression was induced by starvation for methionine and that induction was independent of the promoter and dependent on the leader region terminator. Mutation of conserved primary sequence and structural elements supported a model in which the 5' portion of the leader forms an anti-antiterminator structure, which sequesters sequences required for the formation of an antiterminator, which, in turn, sequesters sequences required for the formation of the terminator; the anti-antiterminator is postulated to be stabilized by the binding of some unknown factor when methionine is available. This set of genes is proposed to form a new regulon controlled by a global termination control system, which we designate the S box system, as most of the genes are involved in sulphur metabolism and biosynthesis of methionine and cysteine.

5' Untranslated Regions↗
PubMed1998

Transcriptional activation of the Bacillus subtilis ackA gene requires sequences upstream of the promoter.

Transcriptional activation of the Bacillus subtilis ackA gene, encoding acetate kinase, was previously shown to require catabolite control protein A (CcpA) and sequences upstream of the ackA promoter. CcpA, which is responsible for catabolite repression of a number of secondary carbon source utilization genes in B. subtilis and other gram-positive bacteria, recognizes a cis-acting consensus sequence, designated cre (catabolite response element), generally located within or downstream of the promoter of the repressed gene. Two sites resembling this sequence are centered at positions -116.5 and -56.5 of the ackA promoter and have been termed cre1 and cre2, respectively. Synthesis of acetate kinase, which is involved in the conversion of acetyl coenzyme A to acetate, is induced when cells are grown in the presence of an easily metabolized carbon source such as glucose. In this study, cre2, the site closer to the promoter, and the region upstream of cre2 were shown to be indispensable for CcpA-dependent transcriptional activation of ackA, whereas cre1 was not required. In addition, insertion of 5 bp between cre2 and the promoter disrupted activation, while 10 bp was tolerated, suggesting face-of-the-helix dependence of the position of cre2 and/or upstream sequences. DNase footprinting experiments demonstrated binding of CcpA in vitro to cre2 but not cre1, consistent with the genetic data. Activation of ackA transcription was blocked in a ptsH1/crh double mutant, suggesting involvement of this pathway in CcpA-mediated transcriptional activation.

Acetate Kinase↗
PubMed1997

Glu-tRNAGln amidotransferase: a novel heterotrimeric enzyme required for correct decoding of glutamine codons during translation.

The three genes, gatC, gatA, and gatB, which constitute the transcriptional unit of the Bacillus subtilis glutamyl-tRNAGln amidotransferase have been cloned. Expression of this transcriptional unit results in the production of a heterotrimeric protein that has been purified to homogeneity. The enzyme furnishes a means for formation of correctly charged Gln-tRNAGln through the transamidation of misacylated Glu-tRNAGln, functionally replacing the lack of glutaminyl-tRNA synthetase activity in Gram-positive eubacteria, cyanobacteria, Archaea, and organelles. Disruption of this operon is lethal. This demonstrates that transamidation is the only pathway to Gln-tRNAGln in B. subtilis and that glutamyl-tRNAGln amidotransferase is a novel and essential component of the translational apparatus.

Acylation↗
PubMed1997

Analysis of cis-acting sequence and structural elements required for antitermination of the Bacillus subtilis tyrS gene.

The Bacillus subtilis tyrS gene belongs to the T box family of aminoacyl-tRNA synthetase and amino acid biosynthesis genes, which are regulated by a common mechanism of transcriptional antitermination. Each gene is induced by specific amino acid limitation; the uncharged cognate tRNA is the effector inducing transcription of the full-length message. The leader regions of the genes in this family share a number of conserved primary sequence and secondary structural elements, the functions of which are unknown. In this study, we examine these regions and report the effects of mutations in several of these elements. In addition, two alternative basepairings in the F box region were found to be necessary for tyrS antitermination.

Bacillus subtilis↗
PubMed1997

The Staphylococcus aureus ileS gene, encoding isoleucyl-tRNA synthetase, is a member of the T-box family.

The Staphylococcus aureus ileS gene, encoding isoleucyl-tRNA synthetase (IleRS), contains a long mRNA leader region. This region exhibits many of the features of the gram-positive synthetase gene family, including the T box and leader region terminator and antiterminator. The terminator was shown to be functional in vivo, and readthrough increased during growth in the presence of mupirocin, an inhibitor of IleRS activity. The S. aureus ileS leader structure includes several critical differences from the other members of the T-box family, suggesting that regulation of this gene in S. aureus may exhibit unique features.

Amino Acid Sequence↗
PubMed1997

Specificity of tRNA-mRNA interactions in Bacillus subtilis tyrS antitermination.

The Bacillus subtilis tyrS gene, encoding tyrosyl-tRNA synthetase, is a member of the T-box family of genes, which are regulated by control of readthrough of a leader region transcriptional terminator. Readthrough is induced by interaction of the cognate uncharged tRNA with the leader; the system responds to decreased tRNA charging, caused by amino acid limitation or insufficient levels of the aminoacyl-tRNA synthetase. Recognition of the cognate tRNA is mediated by pairing of the anticodon of the tRNA with the specifier sequence of the leader, a codon specifying the appropriate amino acid; a second interaction between the acceptor end of the tRNA and an antiterminator structure is also important. Certain switches of the specifier sequence to a new codon result in a switch in the specificity of the amino acid response, while other switches do not. These effects may reflect additional sequence or structural requirements for the mRNA-tRNA interaction. This study includes investigation of the effects of a large number of specifier sequence switches in tyrS and analysis of structural differences between tRNA(Tyr) and tRNA species which interact inefficiently with the tyrS leader to promote antitermination.

Bacillus subtilis↗
PubMed1996

The role of CcpA transcriptional regulator in carbon metabolism in Bacillus subtilis.

The CcpA protein has been identified as a key regulator of carbon metabolism in Bacillus subtilis. CcpA is a DNA binding protein in the LacI/GalR transcriptional repressor family, and genes which respond to CcpA contain common cis-acting target sequences (Ccp boxes). A number of pathways involved in carbon source utilization are repressed by CcpA, while at least one gene which is involved in excretion of excess carbon is activated by CcpA. Genes repressed by CcpA generally contain Ccp boxes within or downstream of the promoter, while ackA, which is activated by CcpA, contains Ccp boxes upstream of the promoter. It therefore appears that CcpA acts globally to direct carbon flow in B. subtilis.

Bacillus subtilis↗
PubMed1996

Control of transcription termination in prokaryotes.

A growing number of genetic systems have been shown to be controlled at the level of premature termination of transcription. Genes in this class contain transcription termination signals in the region upstream of the coding sequence. The activity of these regulatory termination signals is controlled through a variety of mechanisms. These include modification of RNA polymerase to a terminator-resistant, or terminator-prone form, and alterations in the structure of the nascent transcript, to determine whether the stem-loop structure of an intrinsic terminator or an alternate antiterminator is formed. Structural alterations in the transcript can be controlled by the kinetics of translation of the RNA, by binding of specific regulatory proteins, and by mRNA-tRNA interactions. This review describes a number of variations on the termination control theme that have been uncovered in prokaryotes.

Nucleic Acid Conformation↗
PubMed1994

Conservation of a transcription antitermination mechanism in aminoacyl-tRNA synthetase and amino acid biosynthesis genes in gram-positive bacteria.

Most of the aminoacyl-tRNA synthetase genes identified to date in the Gram-positive bacterium Bacillus subtilis have been found to be regulated by readthrough of a transcriptional terminator located in the mRNA leader region, upstream of the start of the coding sequence. All of these leader regions contain a series of conserved structural features, as well as a single codon displayed at a precise position within the structure, which has been shown for tyrS to be responsible for the specificity of the response to limitation for tyrosine. The structural features critical for transcription antitermination in Bacillus aminoacyl-tRNA synthetase genes were found in the B. subtilis ilv-leu biosynthetic operon, the Lactobacillus casei valS gene, the Corynebacterium glutaminicum argS gene, and three amino acid biosynthesis operons in Lactobacillus casei and Lactococcus lactis, suggesting that all of these genes are likely to be regulated by a similar antitermination mechanism. This regulatory system therefore appears to be widespread in Gram-positive bacteria.

Amino Acids↗
PubMed1994

tRNA-directed transcription antitermination.

At least 18 aminoacyl-tRNA synthetase and amino acid biosynthesis genes in several Gram-positive genera appear to be regulated by a common transcription antitermination mechanism. Each gene is induced by limitation for the appropriate amino acid, and not by general amino acid limitation. The mRNA leader regions of these genes exhibit extensive structural conservation. Characterization of the Bacillus subtilis tyrS gene revealed that uncharged tyrosyl-tRNA promotes readthrough of a leader-region terminator; a conformational switch in the leader mRNA between a terminator structure and an antiterminator structure is postulated to mediate antitermination. Two sites of interaction between the tRNA and the leader have been identified by genetic analysis: the tRNA anticodon interacts with a single codon displayed at a precise position in the leader-region structure, and the acceptor end of the tRNA interacts with a side-bulge on the antiterminator.

Amino Acids↗
PubMed1994

Interaction between the acceptor end of tRNA and the T box stimulates antitermination in the Bacillus subtilis tyrS gene: a new role for the discriminator base.

The Bacillus subtilis tyrS gene is a member of a group of gram-positive aminoacyl-tRNA synthetase and amino acid biosynthesis genes which are regulated by transcription antitermination. Each gene in the group is specifically induced by limitation for the appropriate amino acid. This response is mediated by interaction of the cognate tRNA with the mRNA leader region to promote formation of an antiterminator structure. The tRNA interacts with the leader by codon-anticodon pairing at a position designated the specifier sequence which is upstream of the antiterminator. In this study, an additional site of possible contact between the tRNA and the leader was identified through covariation of leader mRNA and tRNA sequences. Mutations in the acceptor end of tRNA(Tyr) could suppress mutations in the side bulge of the antiterminator, in a pattern consistent with base pairing. This base pairing may thereby directly affect the formation and/or function of the antiterminator. The discriminator position of the tRNA, an important identity determinant for a number of tRNAs, including tRNA(Tyr), was shown to act as a second specificity determinant for assuring response to the appropriate tRNA. Furthermore, overproduction of an unchargeable variant of tRNA(Tyr) resulted in antitermination in the absence of limitation for tyrosine, supporting the proposal that uncharged tRNA is the effector in this system.

Bacillus subtilis↗