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PubMed2002

Post-transcriptional control of chloroplast gene expression. Accumulation of stable psaC mRNA is due to downstream RNA cleavages in the ndhD gene.

Intergenic cleavages, intron splicing, and editing of primary transcripts of the plastid ndhH-D operon produce multiple overlapping RNAs, of which the most abundant by far is the monocistronic 400-nucleotide mRNA of psaC (encoding the PsaC protein of photosystem I), in contrast with the low level of transcripts of the six ndh genes. Like other plastid operons containing genes for functionally unrelated proteins, the contrasting accumulation of ndh and psaC transcripts provides a model to investigate the mechanisms of the post-transcriptional control of gene expression, a feature of chloroplast genetic machinery, with a minimum of interference by transcriptional control. In leek (Allium porrum L), the ndhD transcript (which follows the psaC gene and ends the ndhH-D operon) requires C --> U editing to restore its start codon and may be used as a marker for the processing of psaC and ndhD transcripts. By determining the editing state and 5' end sequences of specific transcripts, we demonstrated that stable monocistronic psaC mRNA results from downstream cleavages in the ndhD sequence, which renders non-functional ndhD transcripts as by-products. Alternative psaC-ndhD intergenic cleavages produce complete mRNAs for both genes, but only take place in precursors containing editing-restored ndhD start codons. Hence, post-transcriptional control acts by promoting the ndhD cleavage alternative, which allows the accumulation of psaC mRNA at the expense of ndhD mRNA levels.

Base Sequence↗

PubMed1996

Analysis of the alpha2C-adrenergic receptor gene promoter and its cell-type-specific activity.

As an initial approach to define the regulatory elements and transcriptional factors that account for cell-restricted expression of the alpha2c-adrenergic receptor (AR) gene, we isolated and characterized the receptor gene and identified regions of the gene conferring cell-specific expression. A 4300-nucleotide (nt) fragment of the 5'-flanking region of the rat alpha2c-AR gene was isolated from a genomic library. The genomic sequence contained the uninterrupted sequence of the 5'-untranslated region of a previously isolated alpha2c-AR cDNA clone indicating the lack of introns in the 5' gene segment. RNase protection assays and/or RNA blot analysis indicated the expression of alpha2c-AR mRNA in rat brain but not in kidney or liver, which is consistent with the major expression of this gene in neuronal tissue. The 5' gene segment was used to identify sites of transcriptional initiation and promoter activity by RNase protection assays and transient transfection of reporter gene constructs. With the use of RNA probes progressively upstream of the translational start site, RNase protection assays with rat brain total RNA indicated multiple sites of transcriptional initiation within a approximately 70-nt span (-660 to -730 nt 5' to the translational start codon). The zone of transcriptional initiation was part of a larger GC-rich area of the 5' gene segment that is a characteristic of genes initiating transcripts at multiple sites. The promoter activity of this zone of transcriptional initiation and the influence of gene segments 5' to this area were addressed using chloramphenicol acetyl transferase reporter gene constructs. Transient transfection of reporter gene constructs indicated that a 96-nt gene fragment (-699/-603 relative to the translational start codon) was sufficient to direct transcription in the neuroblastoma X glioma hybrid cell line NG108-15, a cell line expressing the endogenous alpha2c-AR. Promoter activity was not observed in constructs lacking the zone of transcriptional initiation. The promoter segment was inactive when introduced into the rat glioma cell line C6B4, the rat submandibular cell line RSMT-A5, and the rat pancreatic beta cell line RIN-5AH, all of which do not express the endogenous alpha2c-AR gene. Upon incubation with nuclear extracts, a 129-nt fragment encompassing the promoter exhibited a gel mobility shift pattern that was specific for cells expressing the receptor protein and involved a nuclear protein that recognized a Sp1 oligonucleotide. These data indicate that a 96-nt gene promoter segment of the alpha2c-AR gene functions in a cell-type-specific manner.

Animals↗

PubMed1999

Cell transformation results in the loss of the density-dependent translational regulation of the expression of fibroblast growth factor 2 isoforms.

Alternative initiation of translation at three CUG and one AUG start codons leads to the synthesis of four isoforms of fibroblast growth factor 2 (FGF-2) that have distinct intracellular localizations and affect the cell phenotype differently. We show here that the expression of FGF-2 CUG-initiated isoforms decreases in a cell-density-dependent manner in normal human skin fibroblasts (HSFs) concomitantly with the FGF-2 mRNA level. In contrast, CUG-initiated FGF-2 expression is constitutive in SK-HEP-1 cells and in HSFs transformed with SV40 large T antigen. Cell transfection using a plasmid containing the FGF-2 mRNA leader fused to chloramphenicol acetyl transferase demonstrated that up-regulation of the CUG codons depends on cis-elements located in this leader. Furthermore, UV cross-linking experiments revealed a correlation between CUG codons utilization and the binding of several proteins to the mRNA leader. On the basis of the presence of an internal ribosome entry site (IRES) in the FGF-2 mRNA, we used bicistronic vectors to transfect normal and transformed cells. The density-dependent regulation in normal HSFs was cap-dependent, whereas the constitutive CUG-initiated FGF-2 expression in transformed cells occurred essentially by an IRES-dependent mechanism. Unexpectedly, the use of the AUG start codon occurred exclusively by internal entry, which suggests the presence of a second independent IRES in the FGF-2 mRNA that would be constitutive. A study of the eIF-4E levels and of the 4E-BP1 phosphorylation state at increasing cell densities showed a decrease of the eIF-4E level, concomitant with 4E-BP1 dephosphorylation in normal cells but not in transformed cells. These data point out a complex mechanism for the regulation of FGF-2 isoforms expression involving both the cap-dependent and the cap-independent initiation of translation and favor a positive role of CUG-initiated FGF-2 in cellular proliferation and transformation.

Animals↗

PubMed1991

Alternative initiation of translation determines cytoplasmic or nuclear localization of basic fibroblast growth factor.

Three forms of basic fibroblast growth factor (bFGF), initiated at an AUG (18 kDa) and two CUG (21 and 22.5 kDa) start codons, were produced following transfection of COS cells with human hepatoma bFGF cDNA. The subcellular localization of the different forms was investigated directly or by using chimeric genes constructed by fusion of the bFGF and chloramphenicol acetyltransferase open reading frames. The AUG-initiated proteins were cytoplasmic, while the CUG-initiated forms were nuclear. The signal sequence responsible for the nuclear localization of bFGF is contained within 37 amino acid residues between the second CUG and the AUG start codons. Alternative initiation of translation regulates the subcellular localization of bFGF and thus could modulate its role in cell growth and differentiation control.

Amino Acid Sequence↗

PubMed2003

Open reading frame 122 of Helicoverpa armigera single nucleocapsid nucleopolyhedrovirus encodes a novel structural protein of occlusion-derived virions.

Helicoverpa armigera single nucleocapsid nucleopolyhedrovirus (HaSNPV) and its closely related variant H. zea SNPV (HzSNPV) contain 20 open reading frames (ORFs) unique among baculoviruses. In this report, the function of HaSNPV ORF 122 (Ha122) is investigated. Ha122 was transcribed as a polyadenylated transcript from 8 h post-infection in infected H. armigera insect cells. 5'RACE analysis indicated that Ha122 transcription starts predominantly in the consensus major late transcription initiation motif DTAAG, around 47 nt upstream of the putative translation start codon, with a minor start at position -89. Using 3'RACE, the transcription stop site mapped 27 nt downstream of the putative translation stop codon. By Western blot analysis using a chicken-derived polyclonal antibody, the product of Ha122 was found in infected cells to be a 21 kDa protein, close to the theoretical size of 21.6 kDa. The Ha122 protein, when fused to green fluorescent protein, was observed in the nuclei of H. armigera cells but only in conjunction with wild-type HaSNPV infection. The 21 kDa protein was located specifically in the nucleocapsid of occlusion-derived virions (ODVs) and not in that of budded virus. The available data suggest that Ha122 is a functional ORF of HaSNPV and HzSNPV and that the 21 kDa protein is a novel specific component of baculovirus ODVs.

Amino Acid Sequence↗

PubMed1994

Insertional activation of cepA leads to high-level beta-lactamase expression in Bacteroides fragilis clinical isolates.

Bacteroides fragilis is an important opportunistic pathogen of humans and is resistant to many drugs commonly used to treat anaerobic infections, including beta-lactams. A strain set comprised of B. fragilis isolates producing either low or high levels of the endogenous cephalosporinase activity, CepA, has been described previously (M. B. Rogers, A. C. Parker, and C. J. Smith, Antimicrob. Agents Chemother. 37:2391-2400, 1993). Clones containing cepA genes from each of seven representative strains were isolated, and the DNA sequences were determined. Nucleotide sequence comparisons revealed that there were few differences between the cepA coding sequences of the low- and high-activity strains. The cepA coding sequences were cloned into an expression vector, pFD340, and analyzed in a B. fragilis 638 cepA mutant. The results of beta-lactamase assays and ampicillin MICs showed that there was no significant difference in the enzymatic activity of structural genes from the high- or low-activity strains. Comparison of sequences upstream of the cepA coding region revealed that 50 bp prior to the translation start codon, the sequence for high-activity strains change dramatically. This region of the high-activity strains shared extensive homology with IS21, suggesting that an insertion was responsible for the increased expression of cepA in these isolates. Northern (RNA) blot analysis of total RNA by using cepA-specific DNA probes supported the idea that differential cepA expression in low- and high-activity strains was controlled at the level of transcription. However, the insertion did not alter the cepA transcription start site, which occurred 27 bp upstream of the ATG translation start codon in both expression classes. Possible mechanisms of cepA activation are discussed.

Amino Acid Sequence↗

PubMed2006

[Identification and clone of human Alzheimer's disease related gene nicastrin promoter].

OBJECTIVE: To identify the promoter of human nicastrin (NCT) gene, a major component of gamma-secretase which is closely related with pathogenesis of Alzheimer's disease. METHODS: Promoter of human Alzheimer's disease related gene, nicastrin, a 1768 bp fragment was firstly isolated from human genomic DNA by PCR. This fragment's 3 flanking end was 4 bp upstream to the start codon ATG (+1) of the gene. This fragment was used as template, a series of deleted fragments were amplified and constructed to the pGL3-Enhancer plasmid with the artificial designed linkers. The relative activity of their promoter in Hela cells was studied by dual-luciferase assay. RESULTS: The 420 bp fragment showed the strongest activity, and the 237 bp fragment was the minimal fragment in length with activity. CONCLUSION: The promoter of NCT is located at -432/-133 region upstream the translational start codon, while its basal promoter is between -359/-90 that drives the transcription of reporter gene in Hela cells.

Alzheimer Disease↗

PubMed1997

Pre-S2 defective hepatitis B virus infection in patients with fulminant hepatitis.

Controversial data were recently published concerning the association of hepatitis B virus (HBV) variants with fulminant hepatitis (FH). In this study, we first analyzed the complete nucleotide sequences of HBV genomes isolated from serum samples from a surgeon and his mother, who was accidentally infected by the son; both died of FH. The infecting viruses were genetically almost identical in both patients; all the clones examined carried a double nucleotide mutation in the start codon of the pre-S2 region that prevented the synthesis of the corresponding protein. Analyses of different serum samples from the son revealed only wild-type precore sequences in a high viremic serum, whereas hepatitis B e antigen (HBeAg)-defective strains were prevalent when the viremia had decreased. Subsequently, we extended the analysis to the viral genomes isolated from 18 additional patients with acute HBV infection and different clinical behaviors: 3 of 5 patients with FH and without previous liver disease had pre-S2 start codon mutations preventing pre-S2 protein synthesis, whereas none of the 13 control cases had similar genomic rearrangements. Analysis of the precore region showed that viral populations normally producing HBeAg were the only or the prevalent viral strains in all of these cases. In summary, our results support the hypothesis that the pre-S2 protein is not essential for HBV infectivity. They also show that infection by pre-S2-defective virus is frequently associated with FH, indicating that this variant might play a pathogenetic role in cases of acute liver failure. Finally, they suggest that the emergence of HBeAg-defective viruses might be a late event in the course of FH, occurring when HBeAg-producing viruses have been mostly cleared.

Acute Disease↗

PubMed1999

Candidate regulatory sequence elements for cell cycle-dependent transcription in Saccharomyces cerevisiae.

Recent developments in genome-wide transcript monitoring have led to a rapid accumulation of data from gene expression studies. Such projects highlight the need for methods to predict the molecular basis of transcriptional coregulation. A microarray project identified the 420 yeast transcripts whose synthesis displays cell cycle-dependent periodicity. We present here a statistical technique we developed to identify the sequence elements that may be responsible for this cell cycle regulation. Because most gene regulatory sites contain a short string of highly conserved nucleotides, any such strings that are involved in gene regulation will occur frequently in the upstream regions of the genes that they regulate, and rarely in the upstream regions of other genes. Our strategy therefore utilizes statistical procedures to identify short oligomers, five or six nucleotides in length, that are over-represented in upstream regions of genes whose expression peaks at the same phase of the cell cycle. We report, with a high level of confidence, that 9 hexamers and 12 pentamers are over-represented in the upstream regions of genes whose expression peaks at the early G(1), late G(1), S, G(2), or M phase of the cell cycle. Some of these sequence elements show a preference for a particular orientation, and others, through a separate statistical test, for a particular position upstream of the ATG start codon. The finding that the majority of the statistically significant sequence elements are located in late G(1) upstream regions correlates with other experiments that identified the late G(1)/early S boundary as a vital cell cycle control point. Our results highlight the importance of MCB, an element implicated previously in late G(1)/early S gene regulation, as most of the late G(1) oligomers contain the MCB sequence or variations thereof. It is striking that most MCB-like sequences localize to a specific region upstream of the ATG start codon. Additional sequences that we have identified may be important for regulation at other phases of the cell cycle.

5' Untranslated Regions↗

PubMed2000

Antisense oligonucleotide inhibition of angiotensinogen in the brains of rats and sheep.

Phosphorothioated antisense oligodeoxynucleotides (ODNs) that were complementary to various parts of the rat or sheep mRNA encoding angiotensinogen were synthesized by conventional techniques. Their effectiveness as blockers of angiotensinogen synthesis in the brain was tested by bioassay. This involved measuring the effect of centrally administered antisense ODNs on water drinking that occurred in response to intracerebroventricular injection of hog renin. Renin-induced drinking requires brain angiotensinogen for the generation of angiotensin I and then angiotensin II to stimulate thirst. Intracerebroventricular injection of an 18-mer antisense ODN (0.5 microg twice in 24 h) complementary to the 5'-end start codon for rat angiotensinogen mRNA caused a pronounced inhibition of renin-induced drinking. This effect appeared to be specific for this region of the codon because antisense ODNs directed against other regions of rat angiotensinogen mRNA were ineffective, and renin-induced drinking was not inhibited by intracerebroventricular injection of scrambled or mismatched sequences of the effective ODN or by intraperitoneal injection of it. Intracerebroventricular injection of antisense ODN (0.5 microg twice in 24 h) did not inhibit appetite or affect water drinking in response to some other dipsogenic stimuli, thus demonstrating the specificity of its action against renin-induced drinking. By contrast, intracerebroventricular administration of 625 microg of an antisense ODN directed against the corresponding 5'-end start codon region of sheep angiotensinogen mRNA did not inhibit intracerebroventricular renin-induced drinking in sheep. These data show that while intracerebroventricularly administered antisense may be used effectively in rodents, the method is not necessarily applicable in larger mammals.

Angiotensinogen↗

PubMed1999

Localization of promoter elements in the human mu-opioid receptor gene and regulation by DNA methylation.

The regulation of mu-opioid receptor gene expression was investigated using several molecular techniques. Genomic clones containing portions of the human mu-opioid receptor gene were sequenced. 5'-RACE analysis of human brain cDNA confirmed the presence of mRNAs up to -313 from the start codon. As was found for the mouse and rat genes, transcription apparently initiates in the absence of a discernable TATA box. To characterize promoter function, portions of the 5'-flanking region were linked to a reporter gene in transient transfection experiments. Two approximately 50 bp adjacent segments had potent, orientation specific promoter activity. More down-stream segments also had promoter activity. None of the 5'-flanking region constructs showed tissue specificity. The potential role of DNA methylation in preventing ectopic expression was investigated by surveying the methylation state of a CpG rich region straddling the start codon. A neural derived cell line (SH-SY5Y) that expresses the mu-opioid receptor lacked virtually any CpG methylation. In contrast, two neural derived cell lines that do not express the mu-opioid receptor were nearly totally methylated while non-neural cell lines had intermediate levels of CpG methylation. Additional transient transfection experiments revealed that CpG methylation of the 5'-flanking region suppressed reporter gene expression. These results indicate that CpG methylation plays an important role in regulating mu-opioid receptor expression in neural cells; however, no association was found with regulation of expression in non-neural cells.

Animals↗

PubMed2000

rpoS mutants in archival cultures of Salmonella enterica serovar typhimurium.

Long-term survival under limited growth conditions presents bacterial populations with unique environmental challenges. The existence of Salmonella enterica serovar Typhimurium cultures undisturbed in sealed nutrient agar stab vials for 34 to 45 years offered a unique opportunity to examine genetic variability under natural conditions. We have initiated a study of genetic changes in these archival cultures. We chose to start with examination of the rpoS gene since, among gram-negative bacteria, many genes needed for survival are regulated by RpoS, the stationary-phase sigma factor. In each of 27 vials examined, cells had the rpoS start codon UUG instead of the expected AUG of Salmonella and Escherichia coli strains recorded in GenBank. Ten of the 27 had additional mutations in the rpoS gene compared with the X77752 wild-type strain currently recorded in GenBank. The rpoS mutations in the 10 strains included two deletions as well as point mutations that altered amino acid sequences substantially. Since these stored strains were derived from ancestral cells inoculated decades ago and remained undisturbed, it is assumed that the 10 rpoS mutations occurred during storage. Since the remaining 17 sequences were wild type (other than in the start codon), it is obvious that rpoS remained relatively stable during decades of sealed storage.

Amino Acid Substitution↗

PubMed2004

Non-sequencing molecular approaches to identify preS2-defective hepatitis B virus variants proved to be associated with severe liver diseases.

BACKGROUND/AIMS: PreS2-defective hepatitis B virus (HBV) variants may emerge during chronic HBV infection. These variants carry mutation(s) at the ATG-start-codon and/or in-frame deletion into the preS2 genomic region and are commonly detected by sequencing analyses. We evaluated the prevalence of these variants in a large series of chronic HBV infected patients through non-sequencing molecular approaches. METHODS: We examined HBV isolates from 110 HBV carriers: 15 were inactive carriers (IC); 50 had chronic hepatitis (CH); 25 were cirrhotics; 19 had hepatocellular carcinoma (HCC). The entire preS2 genomic region was amplified by PCR technique. The amplicons were processed: (A) through electrophoresis on acrylamide gel to reveal deleted genomes; (B) through electrophoresis on agarose gel after digestion by NlaIII enzyme that cuts the wild ATG-start-codon but not the mutated one. RESULTS: We detected preS2 variants in 56/110 cases (51%). In particular, we found preS2-defective mutants in 2/15 IC, 25/50 CH, 13/26 cirrhotics, and 16/19 HCC. The presence of these variants was thus significantly associated with active infection and liver disease (P<0.002). Moreover, among cases with liver disease preS2-mutants were more prevalent in HCC patients (P<0.02). CONCLUSIONS: Our non-sequencing molecular methods are sensitive and specific, and simplify the identification of all preS2 HBV variant forms. Infection by these variants is significantly associated with active infection and HCC.

Carcinoma, Hepatocellular↗

PubMed1988

Conserved reiterated domains in Clostridium thermocellum endoglucanases are not essential for catalytic activity.

The complete nucleotide sequence of the Clostridium thermocellum celE gene, coding for an endo-beta-1,4-glucanase (endoglucanase E; EGE) with xylan-hydrolysing activity has been determined. The structural gene consists of an open reading frame (ORF) of 2442 bp commencing with a GTG start codon and followed by a TAA stop codon. The nucleotide sequence obtained has been confirmed by comparing the predicted amino acid sequence with that derived by N-terminal amino acid sequencing of the purified protein. The EGE sequence contains a region homologous to the reiterated domain found at the C terminus of other endoglucanases from the same organism. BAL 31 deletions of the structural gene have revealed the extent to which this conserved sequence is necessary for endoglucanase and xylanase activity. A region of DNA, upstream from the structural gene has also been sequenced and a ribosome-binding site and putative promoter sequences have been identified. A second ORF which ends 349 bp 5' to the GTG start codon of the celE gene has also been identified. The encoded product contains a C terminus homologous to other C. thermocellum endoglucanases.

Amino Acid Sequence↗

PubMedDate not supplied

Association of vitamin D receptor-gene (FokI) polymorphism with calcium oxalate nephrolithiasis.

BACKGROUND AND PURPOSE: Formation of kidney stones is still not understood but is hypothesized to be associated with the vitamin D receptor gene (VDR). In order to assess the eventual role of VDR start-codon FokI polymorphism in stone formation, we evaluated the association between calcium stone disease and this polymorphism in a North Indian population. PATIENTS AND METHODS: A control group comprised of 166 healthy individuals (age range 22-58 years) and a group of 138 patients with calcium oxalate stones (age range 21-72 years) were examined. The polymorphism was detected using polymerase chain reaction-based restriction analysis. An unexcisable length of 265 bp (CC) and two fragments of 169 bp and 96 bp (TT) were obtained by FokI restriction digestion. RESULTS: There was a statistically significant difference between the control and patient groups (X2 test, P<0.001) for the genotype of the VDR FokI start-codon polymorphism. The odds ratio (with 95% CI) for the C allele in those at risk of stone disease was 1.654 (1.041, 2.628). The VDR frequency distribution was also statistically significant (P<0.001) in case of male sex. The frequency distribution for this genetic polymorphism was not statistically different in normocalciuric and hypercalciuric stone patients (P=0.355). CONCLUSION: The VDR FokI polymorphism may be a good candidate for a marker for calcium oxalate-stone disease. These findings may contribute a small piece to the understanding of the pathogenesis of urinary calculi.

Adult↗

PubMed2005

Genetic manipulation of two fowlpox virus late transcriptional regulatory elements influences their ability to direct expression of foreign genes.

Fowlpox virus (FWPV) is currently used as a vector to express foreign genes of various poultry and mammalian pathogens. However, due to limited information available about the primary structure of FWPV promoters required for an optimal transcriptional efficiency, the full potential of FWPV as an expression vector has not been completely realized. To dissect such transcriptional regulatory elements at the molecular level, we selected two FWPV promoters dictating contrasting levels of expression of acidic-type inclusion body protein gene (P190) and A15L vaccinia virus homolog of FWPV (P180) for site-directed mutagenesis studies. The transcriptional activity of mutated promoters was analyzed based on their ability to transcribe a reporter gene, lacZ, and translation of the resultant mRNA into functional protein. Replacement of the spacer sequences of P180 with those of P190 resulted in a five-fold increase in mRNA and a 17.6-fold increase in protein over those with its parental promoter, P180. Similarly, replacement of a thymidine after the start codon with guanosine resulted in a 2.3-fold increase in lacZ mRNA and a seven-fold increase in protein. Combining these substitutions in P180SG produced a maximum increase in mRNA and protein of 6.7- and 29.9-fold, respectively, over concentrations with its parental P180 promoter. The promoter activity of P180SG was comparable to that of the strongest natural promoter, P190. The amount of protein per transcript generated by the mutated promoters of P180 increased to at least three times that with the parental P180. In contrast, similar replacements in P190 resulted in a 40-50% reduction in mRNA and protein in all the mutated promoters. We discuss the significance of spacer sequence and the purine after the start codon in the context of a high level of expression.

Animals↗

PubMed2002

Temperature-dependent translation of leaderless and canonical mRNAs in Escherichia coli.

Leaderless mRNAs beginning with a 5'-terminal start codon occur in all biological systems. In this work, we have studied the comparative translational efficiency of leaderless and leadered mRNAs as a function of temperature by in vitro translation competition assays with Escherichia coli extracts. At low temperature (25 degrees C) leaderless mRNAs were found to be translated comparatively better than mRNAs containing an internal canonical ribosome binding site, whereas at high temperature (42 degrees C) the translational efficiency of canonical mRNAs is by far superior to that of leaderless mRNA. The inverse correlation between temperature and translational efficiency characteristic for the two mRNA classes was attributed to structural features of the mRNA(s) and to the reduced stability of the translation initiation complex formed at a 5'-terminal start codon at elevated temperature.

5' Untranslated Regions↗

PubMed2006

Transcriptional and translational control of the Salmonella fliC gene.

The flagellin gene fliC encodes the major component of the flagellum in Salmonella enterica serovar Typhimurium. This study reports the identification of a signal within the 5' untranslated region (5'UTR) of the fliC transcript required for the efficient expression and assembly of FliC into the growing flagellar structure. Primer extension mapping determined the transcription start site of the fliC flagellin gene to be 62 bases upstream of the AUG start codon. Using tetA-fliC operon fusions, we show that the entire 62-base 5'UTR region of fliC was required for sufficient fliC mRNA translation to allow normal FliC flagellin assembly, suggesting that translation might be coupled to assembly. To identify sequence that might couple fliC mRNA translation to FliC secretion, the 5' end of the chromosomal fliC gene was mutagenized by PCR-directed mutagenesis. Single base sequences important for fliC-dependent transcription, translation, and motility were identified by using fliC-lacZ transcriptional and translational reporter constructs. Transcription-specific mutants identified the -10 and -35 regions of the consensus flagellar class 3 gene promoter. Single base changes defective in translation were located in three regions: the AUG start codon, the presumed ribosomal binding site region, and a region near the very 5' end of the fliC mRNA that corresponded to a potential stem-loop structure in the 5'UTR. Motility-specific mutants resulted from base substitutions only in the fliC-coding region. The results suggest that fliC mRNA translation is not coupled to FliC secretion by the flagellar type III secretion system.

5' Untranslated Regions↗