Rho-independent Termination Site Research Articles

Bacteriophage P4 sut1: a mutation suppressing transcription termination

In the Escherichia coli satellite phage P4, transcription starting from PLE is prevalently controlled via premature termination at several termination sites. We identified a spontaneous mutation, P4 sut1 (suppression of termination), in the natural stop codon of P4 orf151 that, by elongating translation, suppresses transcription termination at the downstream t151 site. Both the translational and the transcriptional profile of P4 sut1 differed from those of P4 wild-type. First of all, P4 sut1 did not express Orf151, but a higher molecular mass protein, compatible with the 303 codon open reading frame generated by the fusion of orf151, cnr and the intervening 138 nt. Moreover, after infection of E. coli, the mutant expressed a very low amount of the 1.3 and 1.7 kb transcripts originating at PLE and PLL promoters, respectively, and terminating at the intracistronic t151 site, whereas correspondingly higher amounts of the 4.1 and 4.5 kb RNAs arising from the same promoters and covering the entire operon were detected. Thus the sut1 mutation converts a natural stop codon into a sense codon, suppresses a natural intracistronic termination site and leads to overexpression of the downstream cnr and alpha genes. This correlates with the inability of P4 sut1 to propagate in the plasmid state. By cloning different P4 DNA fragments, we mapped the t151 transcription termination site within the 7633-7361 region between orf151 and gene cnr. A potential stem-loop structure, resembling the structure of a Rho-independent termination site, was predicted by mfold sequence analysis at 7414-7385.

RNA-structural Mimicry in Escherichia coli Ribosomal Protein L4-dependent Regulation of the S10 Operon

Ribosomal protein L4 regulates the 11-gene S10 operon in Escherichia coli by acting, in concert with transcription factor NusA, to cause premature transcription termination at a Rho-independent termination site in the leader sequence. This process presumably involves L4 interaction with the leader mRNA. Here, we report direct, specific, and independent binding of ribosomal protein L4 to the S10 mRNA leader in vitro. Most of the binding energy is contributed by a small hairpin structure within the leader region, but a 64-nucleotide sequence is required for the bona fide interaction. Binding to the S10 leader mRNA is competed by the 23 S rRNA L4 binding site. Although the secondary structures of the mRNA and rRNA binding sites appear different, phosphorothioate footprinting of the L4-RNA complexes reveals close structural similarity in three dimensions. Mutational analysis of the mRNA binding site is compatible with the structural model. In vitro binding of L4 induces structural changes of the S10 leader RNA, providing a first clue for how protein L4 may provoke transcription termination.

Organization of ribosomal RNA genes from a Loofah witches' broom phytoplasma.

Using the technique of integrative mapping with three vectors carrying chromosomal rDNA sequences, one of two rRNA operons of loofah witches' broom (LfWB) phytoplasma was constructed. This is the first complete rRNA operon of a phytoplasma to be reported. The operon has a context of 5'-16S-23S-5S-3' with a tRNA(Ile) gene in the ITS and tRNA(Val) and tRNA(Asn) genes downstream from the 5S rRNA gene. Although the other operon has not been cloned, the DNA sequence of a PCR-amplified product shows that it has no tRNA(Ile) gene in the ITS region. The complete nucleotide sequences of 16S, 23S, and 5S rDNA are 1538, 2864, and 113 bp, respectively. Five -10-like sequences, but no -35 sequences, were found within a 494-bp leader region. There was a TG dinucleotide two nucleotides upstream from each -10-like sequence. The existence of a TG dinucleotide at this position has been reported to enhance the efficiency of a promoter without a -35 region. The regions immediately flanking the 5' and 3' ends of 16S and 23S rDNA can form long basepaired stems that contain sites for processing by RNase III. No obvious sequence for a rho-dependent or rho-independent termination site was found downstream from the tRNA(Asn) gene. The transcription may stop within a pyrimidine-rich region, as has been reported for several polypeptide-encoding genes and rRNA operons of archaeobacteria. The presence of the tRNA genes downstream from the 5S rRNA gene in the rRNA operon of LfWB phytoplasma further supports the hypothesis that phytoplasmas are phylogenetically closer to acholeplasmas than to mycoplasmas. The phylogenetic relatedness of LfWB phytoplasma to other phytoplasmas is discussed on the basis of the nucleotide sequence of rRNA genes and ITS.

An operon of Bacillus subtilis motility genes transcribed by the sigma D form of RNA polymerase

Two genes controlling motility functions in Bacillus subtilis were identified by DNA sequence analysis of a chromosomal fragment containing a strong promoter for sigma D RNA polymerase. Previous studies had shown that this sigma D-dependent promoter controls synthesis of a 1.6-kb transcript in vivo and in vitro. Sequence analysis revealed that the 1.6-kb transcript contains two open reading frames coding for protein sequences homologous to the Escherichia coli motA and motB gene products, respectively, and ends in a rho-independent termination site. Direct evidence linking these genes to motility functions in B. subtilis was obtained by precise localization by polymerase chain reaction of Tn917 transposon insertion mutations of Mot- strains, isolated by Zuberi et al. (A. R. Zuberi, C. Ying, H. M. Parker, and G. W. Ordal, J. Bacteriol. 172:6841-6848, 1990), to within this mot. operon. Replacement of each wild-type gene by in-frame deletion mutations yielded strains possessing paralyzed flagella and confirmed that both motA and motB are required for the motility of B. subtilis. These current findings support our earlier suggestions that sigma D in B. subtilis plays a central role in the control of gene expression for flagellar assembly, chemotaxis, and motility functions. Sigma F, the enteric homolog of sigma D, controls similar functions in E. coli and Salmonella typhimurium, and these factors appear to be representative of a family of factors implicated in flagellar synthesis in many bacterial species, which we propose to designate the sigma 28 family.

Overlapping Transcription and Termination of the Convergent ilvA and ilvY Genes of Escherichia coli

The ilvY gene of Escherichia coli is transcribed in a direction opposite to the ilvGMEDA operon, and the translational stop codons of the ilvA and ilvY genes are only 52 base pairs (bp) apart. We have employed galK transcriptional analyses, in vitro transcription assays, and S1 nuclease mapping to show that the converging transcripts of the ilvGMEDA operon and the ilvY gene overlap. The ilvGMEDA transcript terminates at two sites in the distal amino acid coding region of the ilvY gene: a rho-independent termination site 116 bp downstream of the ilvA translational stop codon, ilvA t, and a 62-bp-long rho-dependent termination site, ilvA t', beginning 70 bp beyond the ilvA t site. This tt' termination pattern at the distal end of the ilvGMEDA operon is similar to that of the trp operon of E. coli and the leu operon of Salmonella typhimurium. Termination of ilvY transcription occurs over a broad stretch of DNA several hundred base pairs downstream of the ilvY translational stop codon in the middle of the amino acid coding region of the ilvA gene. These experiments demonstrate that the converging transcripts from the ilvA and ilvY genes terminate in the coding region of the opposing gene.

Sequence of the Bacillus subtilis glutamine synthetase gene region

The nucleotide sequence of the glutamine synthetase (GS) region of Bacillus subtilis has been determined and found to contain several unique features. An open reading frame (ORF) upstream of the GS structural gene is part of the same operon as GS and is involved in regulation. Two downstream ORFs are separated from glnA by an apparent Rho-independent termination site. One of the downstream ORFs encodes a very hydrophobic polypeptide and contains its own potential RNA polymerase and ribosome-binding sites. The derived amino acid (aa) sequence of B. subtilis GS is similar to that of several other prokaryotes, especially to the GS of Clostridium acetobutylicum. The B. subtilis and C. acetobutylicum enzymes differ from the others in the lack of a stretch of about 25 aa as well as the presence of extra cysteine residues in a region known to contain regulatory as well as catalytic mutations. The region around the tyrosine residue that is adenylylated in GS from many species is fairly similar in the B. subtilis GS despite its lack of adenylylation.

Sequence of cloned enzyme IIN-acetylglucosamine of the phosphoenolpyruvate:N-acetylglucosamine phosphotransferase system of Escherichia coli.

In Escherichia coli, N-acetylglucosamine (nag) metabolism is joined to glycolysis via three specific enzymes that are the products of the nag operon. The three genes of the operon, nagA, nagB, and nagE, were found to be carried by a colicin plasmid, pLC5-21, from a genomic library of E. coli [Clarke, L., & Carbon, J. (1976) Cell (Cambridge, Mass.) 9,91-99]. The nagE gene that codes for enzyme IIN-acetylglucosamine of the phosphoenolpyruvate:sugar phosphotransferase system (PTS) was sequenced. The nagE sequence is preceded by a catabolite gene activator protein binding site and ends in a putative rho-independent termination site. The amino acid sequence determined from this DNA sequence shows 44% homology to enzymes IIglucose and IIIglucose of the PTS. Enzyme IIN-acetylglucosamine, which has 648 amino acids and a molecular weight of 68,356, contains a histidine at residue 569 which is homologous to the active site of IIIglc. Sequence homologies with enzymes IIglucose, II beta-glucoside, and IIsucrose indicate that residues His-190, His-213, and His-295 of enzyme IInag are also conserved and that His-190 is probably the second active site histidine. Other sequence homologies among these enzymes II suggest that they contain several sequence transpositions. Preliminary models of the enzymes II are proposed.

Transcription termination factor rho mediates simultaneous release of RNA transcripts and DNA template from complexes with Escherichia coli RNA polymerase.

Dissociation of RNA and DNA from Escherichia coli RNA polymerase in transcription complexes prepared with enzyme molecules located within and near a rho-dependent transcription termination region on bacteriophage T7 D111 DNA has been studied using a membrane filter-binding assay. Rho protein with ATP present mediated rapid (half-time approximately 27 s) simultaneous dissociation of about 50% of both RNA and DNA. RNA molecules were preferentially released from enzyme molecules located within the termination region. Rapid release of RNA and DNA depended on a nucleoside triphosphate but did not depend on sigma factor. Pretreatment of complexes with ribonuclease prevented dissociation of DNA. Nearly simultaneous dissociation of both RNA and DNA was also detected after a lag of 3 min when the isolated transcription complexes were incubated with all four ribonucleoside triphosphates in the absence of rho factor. In this case, release presumably occurred at the rho-independent termination site that is 5990 nucleotides downstream from the A1 promoter. Thus, the dissociation of DNA from RNA polymerase at rho-dependent and rho-independent transcription termination sites is coupled with or occurs spontaneously soon after the release of transcripts at both sites.

Transcription termination signal for the cat-86 indicator gene in a Bacillus subtilis promoter-cloning plasmid

Plasmid pPL703 is a promoter-cloning plasmid for Bacillus subtilis consisting of the promoter-less cat-86 gene inserted between the EcoRI and BamHI sites of pUB110. The orientation of cat-86 in pPL703 is opposite to that of two major transcript species that occur within the pUB110 vector portion of pPL703. Therefore, transcripts initiated in cloned promoters which activate cat-86 expression presumably must terminate prior to entering the vector portion of pPL703 to permit stable maintenance of promoter-containing derivatives in host cells. We have identified an apparent Rho-independent transcription terminator 35 bp 3' to the cat-86 coding sequence. A restriction fragment spanning the terminator is 90% efficient in terminating transcription in both B. subtilis and Escherichia coli. The structure of the cat-86 transcription termination site is similar to Rho-independent termination sites identified in E. coli.

In vitro transcription of the supB-E tRNA operon of Escherichia coli. Characterization of transcription products.

The seven tRNA genes clustered in the supB-E region of the Escherichia coli chromosome were transcribed in vitro with purified RNA polymerase, using a restriction fragment from lambda psu degrees 2, a transducing phage carrying the chromosome region, as template. A single major transcript was synthesized, which was about 770 nucleotides long and contained all seven tRNA sequences. The terminal sequences of the transcript were determined and mapped on the DNA sequence of the supB-E region previously determined. The transcription start site is seven base pairs downstream from the Pribnow box sequence, as expected from the DNA sequence analysis and consistent with the findings on the trimeric tRNA precursor (pppG--tRNAMETM-tRNALeu-tRNAGln1) which was detected in an RNase P mutant and shown to be coded for by the supB-E region. Cleavage of the restriction fragment at the -35 region with another restriction endonuclease abolished the template activity of the fragment. Transcription of the supB-E tRNA operon was relatively unaffected by the presence of rho factor. Transcription termination occurs within a region of three bases between positions 770 and 772 from the transcription start site. Immediately upstream from the termination sites, there is a region of 26 nucleotides that could form a stem structure, thereby consistent with the general feature of rho-independent termination sites.

Transcription antitermination by bacteriophage lambda N gene product

In the presence of the lambda N gene product, transcription originating at the prophage p L promoter can extend beyond termination signals and into the neighboring bacterial gal operon. In Escherichia coli rho mutants, N function is still required for p L transcription to enter gal. The requirement for N function in rho mutants is abolished by deletion of the prophage genome between 57.4 and 70.9 lambda fractional lengths. These results suggest: 1. (1) Rho-independent termination sites lie in the p L operon of lambda. 2. (2) Both Rho-independent and Rho-dependent terminators are suppressed by N function. N product must, therefore, be considered as a transcription antitermination factor, rather than as an antagonist of Rho, as previously suggested. Various DNA segments have been interposed between p L and gal and their effect on gal escape synthesis studied. Lambda DNA encoding the 6 S transcript, which ends at a Rho-independent terminator, does not reduce gal escape synthesis. The J-b2 region of the phage chromosome, however, completely blocks p L-promoted gal enzyme synthesis; the block is partially relieved by the rho15 mutation. The introduction of large DNA segments lowers gal escape in both rho + and rho15 mutants. The extent of gal escape is directly proportional to the efficiency of suppression of a prophage Nam mutation, suggesting that N product is not made in excess.

Transcription of bacteriophage φX174 in vitro: Selective initiation with oligonucleotides

Transcription in vitro of φX174 RFI† DNA with Escherichia coli RNA polymerase yields three major RNA species of molecular weights 1·87, 1·60 and 1·34×106, the largest of these corresponding to one round of transcription around the DNA circle. In addition there are seven minor species with molecular weights ranging from 0·07 to 0·84×106 which account for about 10 to 20% of the total incorporation of nucleotides into RNA. When rho factor is present in the reaction mixture, the three largest RNAs are no longer made, and instead RNAs of molecular weight 1·00, 0·70, 0·42, 0·35 and 0·07×106 are synthesized. By initiating synthesis at specific promotor sites with oligonucleotide primers and examining the RNA products on polyacrylamide gels, it has been possible to map all of these RNA species and to establish the existence of shared sites of initiation and termination. Three of the RNAs are shown to be artifacts of the synthesis conditions used. The others result from the combined action of three promotors, two rho-independent termination sites, and two rho-dependent termination sites. RNA molecules initiated from two of the promotors begin with 5′-terminal ATP, from the other promotor with 5′-terminal GTP.

Transcription of bacteriophage T4 DNA by Escherichia coli RNA polymerase in vitro: Identification of some immediate-early and delayed-early genes

Transcription of bacteriophage T4 DNA by Escherichia coli RNA polymerase has been studied in vitro. RNA synthesis was initiated at sigma-specific promotor loci by forming rifampicin-resistant initiation complexes before the onset of polymerization and specific termination accomplished by the addition of rho factor. The RNA synthesized in the presence and absence of rho was analyzed for gene-specific messenger RNA's. The eight genes studied could be grouped into three classes: (1) those which were transcribed in the presence or absence of rho (genes 42 and 30); (2) those which were transcribed only in the absence of rho (genes 43, e, rIIA and rIIB); and (3) those which could not be transcribed in vitro even in the absence of rho (genes 41 and 40). It is concluded that genes 42 and 30 belong to the immediate-early class and genes 43, rIIA and rIIB to the delayed-early class. The transcription of the lysozyme gene in vitro resembles that of the delayed-early genes. The inability to transcribe gene 41 in vitro even in the absence of rho implies a rho-independent termination site between genes 42 and 41 and a positive control element to turn on gene 41.