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PubMed1988

Secondary structure of the leader transcript from the Escherichia coli S10 ribosomal protein operon.

Genetic analysis of the autogenous control of the S10 ribosomal protein operon of Escherichia coli has suggested that the secondary or tertiary structure of the leader transcript is important for this regulation. We have therefore determined the secondary structure of the leader by enzyme digestion and chemical modification. Our results suggest that the 172 base leader exists in two forms, differing only immediately upstream of the Shine-Dalgarno sequence of the first gene. We discuss the possibility that the equilibrium between these alternate structures is important for the L4-mediated regulation of translation of the S10 operon. We have also determined the structure of several mutant transcripts. Correlation of these structures with the regulatory phenotypes suggest that a hairpin about 50 bases upstream of the first gene is essential for the control of translation of the operon. Finally, our results show that a two base substitution in an eight base loop destabilizes the attached stem.

Base Sequence↗

PubMed1989

Euglena gracilis chloroplast ribosomal protein operon: a new chloroplast gene for ribosomal protein L5 and description of a novel organelle intron category designated group III.

We describe the structure (3840 bp) of a novel Euglena gracilis chloroplast ribosomal protein operon that encodes the five genes rpl16-rpl14-rpl5-rps8-rpl36. The gene organization resembles the spc and the 3'-end of the S10 ribosomal protein operons of E. coli. The rpl5 is a new chloroplast gene not previously reported for any chloroplast genome to date and also not described as a nuclear-encoded, chloroplast protein gene. The operon contains at least 7 introns. We present evidence from primer extension analysis of chloroplast RNA for the correct in vivo splicing of five of the introns. Two of the introns within the rps8 gene flank an 8 bp exon, the smallest exon yet characterized in a chloroplast gene. Three introns resemble the classical group II introns of organelle genomes. The remaining 4 introns appear to be unique to the Euglena chloroplast DNA. They are uniform in size (95-109 nt), share common features with each other and are distinct from both group I and group II introns. We designate this new intron category as 'group III'.

Amino Acid Sequence↗

PubMed1990

Gene F of plasmid RSF1010 codes for a low-molecular-weight repressor protein that autoregulates expression of the repAC operon.

The repAC operon of plasmid RSF1010 consists of the genes for proteins E, F, RepA (DNA helicase), and RepC (origin-binding initiator protein) and is transcriptionally initiated by a promoter called P4. We have studied the expression of the repAC operon in vivo by using fusions to the lacZ reporter gene. The results show that the product of the second gene, F, autoregulates the operon by inhibiting transcription from P4. To verify its properties postulated from the in vivo studies and to initiate its biochemical characterization, we have purified the F protein from an overproducing E.coli strain constructed in vitro. Purification was based on a gel retardation assay for detection of P4-specific DNA binding. Subsequent DNase footprinting of the F binding sites showed clear protection around two partially symmetric P4 sequences of 16 bp, each of which matches the symmetric consensus sequence, GCGTGAGTACTCACGC, in at least 13 positions. The native repressor, as judged from gel filtration, velocity sedimentation and crosslinking studies, exists as a dimer in dilute solution; its monomeric subunit, as predicted from DNA sequence and N-terminal protein sequence data, consists of 68 amino acids and has a calculated M tau = 7,673.

Base Sequence↗

PubMed1990

The Euglena gracilis chloroplast rpoB gene. Novel gene organization and transcription of the RNA polymerase subunit operon.

The rpoB gene coding for a beta-like subunit of the chloroplast DNA-dependent RNA polymerase has been located on the chloroplast genome of Euglena gracilis distal to the rrnC ribosomal RNA operon. We have determined 5760 base-pairs of DNA sequence, including 97 bp of the 5S rRNA gene, an intergenic spacer of 1264 bp, the rpoB gene of 4249 bp, 84 bp spacer and 67 bp of the rpoC1 gene. The rpoB gene is of the same polarity as the rRNA operons. The organization of the rpoB and rpoC genes resembles the E. coli rpoB-rpoC and higher plant chloroplast rpoB-rpoC1-rpoC2 operons. The Euglena rpoB gene (1082 codons) encodes a polypeptide with a predicted molecular weight of 124,288. The rpoB gene is interrupted by seven Group III introns of 93, 95, 94, 99, 101, 110 and 99 bp respectively and a Group II intron of 309 bp. All other known rpoB genes lack introns. All the exon-exon junctions were experimentally determined by cDNA cloning and sequencing or direct primer extension RNA sequencing. Transcripts from the rpoB locus were characterized by Northern hybridization. Fully-spliced, monocistronic rpoB mRNA, as well as rpoB-rpoC1 and rpoB1-rpoC1-rpoC2 mRNAs were identified.

Amino Acid Sequence↗

PubMed1991

A novel RNA product of the tyrT operon of Escherichia coli.

The tyrT operon of E. coli and several other tRNA operons of E. coli show striking structural features: they contain repeated sequence units including a 19bp motif resembling the 3' end of the corresponding mature tRNA. A novel RNA, encoded by the repeated sequence of the tyrT operon, was identified. The RNA, characterized by primer extension and Nuclease-S1 analysis, contained 171 nucleotides and terminated with the 19bp motif of the CCA-end of tRNA(1Tyr). The RNA, designated as rtT RNA, is probably released from the primary transcript of tyrT during tRNA processing, it includes the coding capacity for the arginine rich peptide Tpr. Predictions of secondary folding resulted a rather stable RNA structure with a free energy of -44.3kcal/mol. A weak ribosome binding site was found, preceding the second possible AUG initiator codon for Tpr. The comparison of rtT RNA with putative transcripts from the repeated sequences associated with related tRNA genes showed common features with respect to primary structure, arrangement and secondary folding. In E. coli cultures the lag-phase during growth, caused by transient glycine or by isoleucine limitation, was found to be overcome or markedly shortened in the presence of rtT RNA. These and previously reported results suggest a modulatory effect of rtT RNA on stringent response.

Amino Acid Sequence↗

PubMed1975

Specific binding of the first enzyme for histidine biosynthesis to the DNA of histidine operon.

Studies were done to examine direct binding of the first enzyme of the histidine biosynthetic pathway (phosphoribosyltransferase) to 32P-labeled phi80dhis DNA and competition of this binding by unlabeled homologous DNA and by various preparations of unlabeled heterologous DNA, including that from a defective phi80 bacteriophage carrying the histidine operon with a deletion of part of its operator region. Our findings show that phosphoribosyltransferase binds specifically to site in or near the regulatory region of the histidine operon. The stability of the complex formed by interaction of the enzyme with the DNA was markedly decreased by the substrates of the enzyme and was slightly increased by the allosteric inhibitor, histidine. These findings are consistent with previous data that indicate that phosphoribosyltransferase plays a role in regulating expression of the histidine operon.

ATP Phosphoribosyltransferase↗

PubMed1992

The promoter of the tgt/sec operon in Escherichia coli is preceded by an upstream activation sequence that contains a high affinity FIS binding site.

The tgt/sec operon in E. coli consists of five genes: queA, tgt, ORF12, secD, and secF. QueA and Tgt participate in the biosynthesis of the hypermodified t-RNA nucleoside Queuosine, whereas SecD and SecF are involved in protein secretion. Examination of the promoter region of the operon showed structural similarity to promoter regions of the rrn-operons. An upstream activation sequence (UAS) containing a potential binding site for the factor of inversion stimulation (FIS) was found. Gel retardation assays and DNaseI footprinting indicated, that FIS binds specifically and with high affinity to a site centred at position -58. Binding of FIS caused bending of the DNA, as deduced from circular permutation analysis. Various 5' deletion mutants of the promoter region were constructed and fused to a lacZ reporter gene to determine the influence of the UAS element on the promoter strength. An approximately two-fold activation of the promoter by the UAS element was observed.

Base Sequence↗

PubMed1999

Caenorhabditis elegans mRNAs that encode a protein similar to ADARs derive from an operon containing six genes.

The Caenorhabditis elegans T20H4.4 open reading frame (GenBank accession no. U00037) predicted by Genefinder encodes a 367 amino acid protein that is 32-35% identical to the C-terminal domain of adenosine deaminases that act on RNA. We show that T20H4.4 cDNAs (GenBank accession no. AF051275) encode a larger 495 amino acid protein that is extended at its N-terminus to include a single double-stranded RNA-binding motif, and that T20H4.4 occupies the second position in a six-gene operon (5'-T20H4.5, T20H4.4, R151.8A, R151.8B, R151.7, R151.6-3'). Ten different spliced-leader (SL) sequences were found attached to T20H4.4 mRNAs, including SL1, SL2 and eight SL2-like leaders that include two new variants. Characterization of cDNAs derived from all six genes confirmed the essential features of C.elegans operons: intercistronic distances in the range of 104-257 nt between the upstream polyadenylation sites and the downstream trans -splice sites; SL2, or SL2-like leaders, attached to the downstream mRNAs. Polycistronic mRNA fragments revealed a 5'-untranslated region (5'-UTR) >705 nt. The 5'-UTR is removed in mature mRNAs from the first gene (T20H4.5) and replaced primarily by SL1, and to a lesser extent by SL2. Our study provides new information regarding operons and how they are processed.

5' Untranslated Regions↗

PubMed2001

rrndb: the Ribosomal RNA Operon Copy Number Database.

The Ribosomal RNA Operon Copy Number Database (rrndb) is an Internet-accessible database containing annotated information on rRNA operon copy number among prokaryotes. Gene redundancy is uncommon in prokaryotic genomes, yet the rRNA genes can vary from one to as many as 15 copies. Despite the widespread use of 16S rRNA gene sequences for identification of prokaryotes, information on the number and sequence of individual rRNA genes in a genome is not readily accessible. In an attempt to understand the evolutionary implications of rRNA operon redundancy, we have created a phylogenetically arranged report on rRNA gene copy number for a diverse collection of prokaryotic microorganisms. Each entry (organism) in the rrndb contains detailed information linked directly to external websites including the Ribosomal Database Project, GenBank, PubMed and several culture collections. Data contained in the rrndb will be valuable to researchers investigating microbial ecology and evolution using 16S rRNA gene sequences. The rrndb web site is directly accessible on the WWW at http://rrndb.cme. msu.edu.

Databases, Factual↗

PubMed2026

Selective targeting of a histone-like silencer Sfx to the R6K conjugal transfer operon.

Conjugative plasmids drive bacterial evolution and antibiotic resistance spread, yet their gene expression must be silenced to protect the host. A histone-like protein H-NS represses many mobile and sedentary xenogenes but fails to silence the conjugal transfer vir operon of R6K, a prototype IncX plasmid. Instead, R6K encodes its own H-NS homolog, Sfx, to repress the vir operon. Here, we show that, unlike other plasmid silencers that target promoters, Sfx cooperates with Rho factor to arrest transcription elongation. ChIP-seq reveals that Sfx and H-NS share similar DNA motifs and a preference for negative supercoiling, but occupy reciprocal genomic niches; Sfx is enriched on the R6K vir operon despite weak chromosomal binding, whereas H-NS displays the opposite preference. We show that Sfx binding to vir DNA critically depends on DNA topology and hypothesize that its selective targeting to R6K is mediated by Sfx-vir interactions and phase separation. Our results suggest that Sfx phase separates with R6K to ensure its preferential recruitment to the plasmid DNA and forms stable bridged nucleoprotein filaments that are impermeable to competitors such as H-NS. These findings reveal how histone-like proteins can partition the genome into distinct regulatory niches, a strategy likely mirrored across all life.

Operon↗

PubMed2001

Genetic analysis of the isc operon in Escherichia coli involved in the biogenesis of cellular iron-sulfur proteins.

The iron-sulfur (Fe-S) cluster, the nonheme-iron cofactor essential for the activity of many proteins, is incorporated into target proteins with the aid of complex machinery. In bacteria, several proteins encoded by the iscRSUA-hscBA-fdx-ORF3 cluster (isc operon) have been proposed to execute crucial tasks in the assembly of Fe-S clusters. To elucidate the in vivo function, we have undertaken a systematic mutational analysis of the genes in the Escherichia coli isc operon. In all functional tests, i.e. growth rate, nutritional requirements and activities of Fe-S enzymes, the inactivation of the iscS gene elicited the most drastic alteration. Strains with mutations in the iscU, hscB, hscA, and fdx genes also exhibited conspicuous phenotypical consequences almost identical to one another. The effect of the inactivation of iscA was small but appreciable on Fe-S enzymes. In contrast, mutants with inactivated iscR or ORF3 showed virtually no differences from wild-type cells. The requirement of iscSUA-hscBA-fdx for the assembly of Fe-S clusters was further confirmed by complementation experiments using a mutant strain in which the entire isc operon was deleted. Our findings support the conclusion that IscS, via cysteine desulfurase activity, provides the sulfur that is subsequently incorporated into Fe-S clusters by assembler machinery comprising of the iscUA-hscBA-fdx gene products. The results presented here indicate crucial roles for IscU, HscB, HscA, and Fdx as central components of the assembler machinery and also provide evidence for interactions among them.

Amino Acids↗

PubMed1983

Nucleotide sequence of the regulatory region of malB operons in E. coli.

The nucleotide sequence of a cloned section of the Escherichia coli chromosome containing the promoter regions of the malB divergent operons was determined. The region of the proximal gene, malE of the malEFG operon, was identified on the basis of the known amino acid sequence of the precursor molecule of maltose-binding protein. The region of malK, the proximal gene of the malKlamB operon, was deduced from the observation that a cloned segment contains an amino-terminal portion of the malK gene. The non-coding region between malE and malK is 299 base pairs long and contains two long GC clusters. Another feature of this region that may be related to the regulation of gene expression is the presence of two palindromic structures between the GC clusters. The DNA regions binding to cyclic AMP binding protein were determined by a method using polyacrylamide gel electrophoresis. The sites are thought to be located close to GC clusters.

Base Sequence↗

PubMed1998

Activation of the bgl operon by adaptive mutation.

In growing Escherichia coli K12 cells, the cryptic bgl operon is activated 98% of the time by insertions of IS1 or IS5 into the control region, designated bglR. The activated bgl operon permits utilization of the beta-glucoside sugar arbutin as a sole carbon and energy source. The bgl operon is also activated by late-occurring mutations during prolonged selection on arbutin. The late-occurring mutations that occurred during prolonged carbon starvation in the presence of arbutin were "adaptive mutations" because they were specific to the presence of arbutin, and they did not occur during prolonged starvation in the absence of arbutin. The spectrum of late-arising mutations differed from that of early-arising, growth-dependent mutations in that 20% of the late-arising mutants resulted from mutations at the hns locus. This provides the first direct evidence for adaptive mutagenesis mediated by the insertion of IS elements. Because no special genetic background is required to select Bgl+ mutants, this affords the opportunity to study IS-element-mediated adaptive mutagenesis in a variety of genetic backgrounds, including the backgrounds of natural isolates of E. coli.

Adaptation, Physiological↗

PubMed1996

Expression of the puf operon in an aerobic photosynthetic bacterium, Roseobacter denitrificans.

The effects of oxygen and light on the expression of the puf operon were investigated in Roseobacter denitrificans in a comparison with those in Rhodobacter sphaeroides. In darkness, the levels of the total puf mRNA in Ros. denitrificans were about 1.3 times those in Rb. sphaeroides at low concentrations of oxygen, reflecting the accumulation of bacteriochlorophyll and carotenoids. The oxygen tension, up to 94% saturation of dissolved oxygen, did not affect the levels of the total puf transcripts in Ros. denitrificans, whereas those in Rb. sphaeroides were reduced to 55% of the maximum level even at 50% saturation. Four puf-specific transcripts were detected: a 0.5-kb transcript was the most abundant; 1.2-kb and 1.9-kb transcripts accumulated at low levels; and a 3.5-kb transcript accumulated at very low levels under all conditions tested. The levels of the individual transcripts were barely affected by molecular oxygen. An S-1 nuclease protection assay revealed that the 0.5-kb transcript encoded the LHI-alpha and LHI-beta subunits (pufBA), the 1.2-kb transcript encoded puf-BA and part of pufL, and the 1.9-kb transcript encoded pufBAL and part of pufM. It was not clear whether the 3.5-kb transcript encoded the entire pufBALM and the gene for the polypeptide moiety of cytochrome c. The difference in levels between the 0.5-kb transcript and the other transcripts (1.2 kb, 1.9 kb, and 3.5 kb) was presumed to be due to the presence of several stem-loop structures at the 3' terminus of the 0.5-kb transcript which acted as terminators of transcription and, possibly, as protection against nucleolytic digestion. Light inhibited the expression of the puf operon in Ros. denitrificans more effectively than that in Rb. sphaeroides. The insensitivity to oxygen, as well as the sensitivity to light, of the expression of the puf operon in Ros. denitrificans, which was different from that in Rb. sphaeroides, seemed to represent a mode of adaptation that allowed the former cells to avoid photodynamic damage by light under highly aerobic conditions.

Bacteria↗

PubMed2001

A phycocyanin-deficient mutant of synechocystis PCC 6714 with a single-base substitution upstream of the cpc operon.

The structure and expression of the cpc operon encoding phycocyanin subunits and linker polypeptides in a phycocyanin-deficient mutant (PD-1) and the wild-type of Synechocystis PCC 6714 were analyzed. The results of sequence and Northern blot analyses of the wild type indicate that the cpc operon consists of cpcB, cpcA, cpcC1, cpcC2 and cpcD, in that order. The levels of the transcripts in PD-1 were one-tenth to one-sixth as high as those in the wild type. In the PD-1 genome, a single-base substitution of C for T has occurred at base 259 upstream of the translational initiation codon of cpcB (at three bases downstream of the putative -10 region). To evaluate the in vivo transcription activities of these promoters in a cyanobacterium, we constructed vectors for the transformation of Synechococcus PCC7942, pANY1 and pANY2, which contain the upstream region of cpcB of the wild type (pANY1) or PD-1 (pANY2) and the promoter-less luxAB fusion. The bioluminescence of the transformants with pANY2 was one-tenth to one-sixth as high as that with pANY1. The coincidence of the results of Northern analysis and the promoter assay shows that the phycocyanin deficiency of PD-1 is due to the single-base substitution in the upstream region of the cpc operon.

Base Sequence↗

PubMed2006

The ntrPR operon of Sinorhizobium meliloti is organized and functions as a toxin-antitoxin module.

The chromosomal ntrPR operon of Sinorhizobium meliloti encodes a protein pair that forms a toxin-antitoxin (TA) module, the first characterized functional TA system in Rhizobiaceae. Similarly to other bacterial TA systems, the toxin gene ntrR is preceded by and partially overlaps with the antitoxin gene ntrP. Based on protein homologies, the ntrPR operon belongs to the vapBC family of TA systems. The operon is negatively autoregulated by the NtrPNtrR complex. Promoter binding by NtrP is weak; stable complex formation also requires the presence of NtrR. The N-terminal part of NtrP is responsible for the interaction with promoter DNA, whereas the C-terminal part is required for protein-protein interactions. In the promoter region, a direct repeat sequence was identified as the binding site of the NtrPNtrR complex. NtrR expression resulted in the inhibition of cell growth and colony formation; this effect was counteracted by the presence of the antitoxin NtrP. These results and our earlier observations demonstrating a less effective downregulation of a wide range of symbiotic and metabolic functions in the ntrR mutant under microoxic conditions and an increased symbiotic efficiency with the host plant alfalfa suggest that the ntrPR module contributes to adjusting metabolic levels under symbiosis and other stressful conditions.

Anti-Bacterial Agents↗

PubMedDate not supplied

Transcription of the octopine catabolism operon of the Agrobacterium tumor-inducing plasmid pTiA6 is activated by a LysR-type regulatory protein.

Agrobacterium tumefaciens incites crown gall tumors on plant hosts by conjugally transferring a discrete fragment of oncogenic DNA. In addition to oncogenes, the transferred DNA contains genes that direct the synthesis and exudation of opines, which are used as nutrients by the bacteria. The bacterium contains one or more operons of Ti plasmid-encoded genes that are required for the internalization and utilization of opines, and transcription of these catabolic genes is induced by cognate opines. Here we localize the gene required for regulated expression of the octopine degradative operon of the pTiA6 plasmid to a 2-kb fragment of Ti plasmid DNA. The protein encoded by this DNA positively regulates the transcription of the catabolic operon in the presence of octopine. In addition, it negatively regulates its own gene in the presence or absence of octopine. The sequence of this gene was determined and analysis of the inferred protein sequence indicates that the gene encodes a member of the LysR family of prokaryotic transcriptional regulatory proteins.

Agrobacterium tumefaciens↗

PubMedDate not supplied

Characterization of the hrpJ and hrpU operons of Pseudomonas syringae pv. syringae Pss61: similarity with components of enteric bacteria involved in flagellar biogenesis and demonstration of their role in HarpinPss secretion.

The hrp/hrmA gene cluster of Pseudomonas syringae pv. syringae Pss61 has been shown to form a minimum genetic unit sufficient to enable nonpathogenic bacteria, such as Escherichia coli, to elicit the hypersensitive response associated with disease resistance. The biochemical functions of most of these genes have not been established. The nucleotide sequence of a 4.3-kb SstI-BglII fragment carrying hrp apparent translational units V, VI, and VII revealed one partial open reading frame (ORF) and five complete ORFs producing 35,126-, 48,866-, 17,308-, 20,482-, and 26,364-Da gene products (hrpJ3, J4, J5, U1, U2, respectively). The production of these proteins was confirmed by using T7 RNA polymerase-directed expression. The partial ORF was found to be identical to the C terminus of HrpJ2. The absence of apparent transcriptional terminators and promoters between hrpI (hrpJ2), hrpJ3, hrpJ4, and hrpJ5 together with the observation that the HrpL-dependent hrpJ promoter directs expression of hrpJ3-J5 indicates that these genes form a single operon controlled by the HrpL-dependent hrpJ promoter. A second HrpL-dependent promoter consensus sequence was also identified upstream of hrpU1 and demonstrated to function as a HrpL-dependent promoter, thus indicating that hrpU1, hrpU2, and additional downstream genes may be part of a second operon. The deduced product of hrpJ3 exhibits similarity to FliG of Salmonella typhimurium, a cytoplasmic protein that regulates flagellar rotation and biogenesis. HrpJ4 shares extensive similarity with the FliI family of ATPase-like proteins and retains the known functional domains conserved among this family of proteins. HrpJ5 has properties similar to the S. typhimurium FliJ. Neither HrpU1 nor HrpU2 exhibit significant similarity to known proteins. Secretion of HarpinPss by E. coli MC4100 transformants carrying pHIR11::TnphoA derivatives was blocked in hrpJ4, J5, and U2 mutants. In view of the previously reported similarity of HrpJ2 to the LcrD super-family that includes FlhA, these results predict that the gene products of the hrpJ and hrpU operons form an inner membrane complex for translocation of proteins similar to that used by the flagellar biogenesis system of S. typhimurium.

Amino Acid Sequence↗