Rho-independent Termination Signal Research Articles

The ribonuclease-III-processing site near the 5' end of an RNA precursor of bacteriophage T4 and its effect on termination.

Infection of RNase III- (rnc) Escherichia coli cells with bacteriophage T4 delta 27, a deletion mutant missing seven out of the ten genes in the tRNA transcription unit, results in the accumulation of a tRNA precursor (10.5-S RNA) that contains the sequences of tRNAGln, tRNALeu and species 1 RNA [Pragai and Apirion (1981) J. Mol. Biol. 153, 619-630]. In vitro studies, using partially purified RNase III or cell extracts and 10.5-S RNA as substrate, have revealed a cleavage site at the 5' side of the molecule. A computerized secondary structure suggests that the RNase III cleavage site can be placed in a small bulge which could be part of a duplex structure and is adjacent to A-A-G and its complementary sequence U-U-U in the same relative relationships found for most RNase III cleavage sites were the adjacent sequences are (A-A-G/U-U-C). Under normal processing conditions (presence of RNase III) the 3' end of the processed intermediate precursors, 10.1-S and p2Sp1 RNAs, is C-U-U-(U1-2)-UOH, which is determined by a stem and loop structure that could serve as a rho-independent termination signal site. However, in the absence of RNase III, the accumulated 10.5-S precursor RNA does not terminate at the same site and its 3' end is shifted a few nucleotides downstream. Thus, RNase III, besides playing a role in processing of 10.5-S RNA, also affects the termination of that molecule, even though both sites, the RNase III cleavage site and the termination site, are about 390 nucleotides apart.

“ADP Sulfurylase” from Thiobacillus denitrificansIs an Adenylylsulfate:Phosphate Adenylyltransferase and Belongs to a New Family of Nucleotidyltransferases

During AMP-dependent sulfite oxidation by some sulfur bacteria, the liberation of sulfate from adenosine-5'-phosphosulfate (APS) is catalyzed by APS:phosphate adenylyltransferase (APAT). Here we report the first biochemical and genetic characterization of APAT. We isolated this enzyme from the chemolithoautotroph Thiobacillus denitrificans and cloned the corresponding gene. The enzyme is homodimeric with 41,387-Da subunits and exhibits a specific activity of 2100 micromol min(-1) mg(-1). The K(m) values are K(m(APS)) = 300 microM and K(m(P(i))) = 12 mM. Catalysis occurs by a ping-pong mechanism with a covalently bound AMP as reaction intermediate. The arsenolysis of APS, but not of ADP, CDP, GDP, UDP, or IDP, is also catalyzed, indicating a specific and unidirectional function. The former enzyme name ADP-sulfurylase implies that the reverse reaction is catalyzed; therefore, this name should not be used any longer. Histidine modification of APAT results in complete inactivation that can be suppressed by substrate addition. APAT is highly similar to galactose-1-phosphate uridylyltransferase and also related to Ap(4)A phosphorylase. Active site residues of galactose-1-phosphate uridylyltransferase are conserved in APAT and Ap(4)A phosphorylase, suggesting a histidine as the nucleotide-binding residue in all three enzymes, which together form a new family of nucleotidyltransferases.

Nucleotide sequence of the gene presumably encoding the adsorption protein of filamentous phage φLf

A 1170-nucleotide fragment of φLf DNA was sequenced. This fragment contains an open reading frame, ORF367, encoding a protein of 367 amino acids (aa) (36710 Da). ORF367 is located downstream from the gene encoding the major coat protein (gVIIIp) and a Rho-independent termination signal. Sequence analysis revealed that the gene product has a Gly-rich domain (70 aa) at the center and a hydrophobic region (26 aa) at the C terminus. These structural features suggest that ORF367 may encode the adsorption protein of φLf.

Characterization and expression of a gene encoding serine tRNA 5 from Escherichia coli

The genes for translational components frequently are located together on the Escherichia coli genome. We have reported previously that the gene for a serine tRNA lies directly downstream from infA, the gene encoding initiation factor IF1. Here we characterize this tRNA gene, named serW. The serW gene expresses a minor form of serine tRNA GGA which recognizes the most frequently used serine codons, UCC and UCU. Two promoters were identified by S1 nuclease mapping: P1, which lies about 72 bp upstream from the structural gene; and P2, which lies about 35 bp upstream. Expression from P1 and P2 is comparable under conditions of rapid growth. The P2 promoter is followed by a GC-rich element characteristic of promoters regulated by ppGpp. A putative hairpin structure followed by a stretch of U residues about 25 nucleotides following the mature tRNA sequence resembles a rho-independent termination signal. The upstream gene, infA, is followed by a transcriptional terminator, but S1 mapping shows considerable readthrough. Thus serW expression appears to rely both on its own promoters and on promoters further upstream. The downstream gene, encoding an unidentified protein of about 100 kDa, is expressed in the opposite orientation and also is followed by a termination signal. Therefore serW is expressed both as a monocistronic gene and in combination with infA.

Insertion (sufB) in the anticodon loop or base substitution (sufC) in the anticodon stem of tRNA(Pro)2 from Salmonella typhimurium induces suppression of frameshift mutations.

The dominant +1 frameshift suppressors sufA6, sufB1 and sufB2, in Salmonella typhimurium act at runs of C and affect tRNA(Pro)1, tRNA(Pro)2 and tRNA(Pro)2, respectively. A recessive +1 frameshift suppressor, sufC, has a similar suppressor specificity (Riddle, D.L., and Roth, J.R., Mol. Biol. 66, 483 and 495, 1972). We show that sufC strains harbour two frameshift suppressors of which one, sufX201, is allelic to sufB. We cloned the sufB+ wild type allele and by recombination in vivo the mutations sufB1, sufB2 and sufX201. Determination of the DNA sequence revealed that the sufB1 and sufB2 mutations result in an extra G in the anticodon loop of the minor tRNA(Pro)2. The sufX201 mutation results in a base substitution (G43 to A43) in the anticodon stem of this tRNA. Although the sufB1 and sufB2 mutations were earlier shown to be dominant, the sufB+ wild type allele on multi copy plasmid inhibited the chromosomal sufB1, sufB2 and sufX201 mediated frameshift suppression but not that mediated by the dominant sufA6 mutation. These results are discussed in view of the possible coding specificity of these mutated tRNAs. The DNA sequence showed a potential consensus promoter sequence upstream of the structural gene for tRNA(Pro)2 and downstream a dyad symmetrical structure followed by a T cluster, a possible rho-independent termination signal. The Salmonella tRNA(Pro)2 gene is identical to the Escherichia coli counterpart reported by Komine, Y. et al. (J. Mol. Biol. 212, 579-598, 1990). While the 5' flanking sequence similarity between the two species is about 83%, the similarity of the 3' flanking sequence is only 42%. Still, the Salmonella tRNA(Pro)2 gene has a rho-independent transcriptional termination signal similar to the one present in E. coli tRNA(Pro)2 gene.

Delineation of the transcriptional boundaries of the lux operon of Vibrio harveyi demonstrates the presence of two new lux genes.

The 5' and 3' ends of the lux mRNA of Vibrio harveyi, which extends over 8 kilobases, have been mapped, and two new genes, luxG and luxH, were identified at the 3' end of the lux operon. Both S1 nuclease and primer extension mapping demonstrated that the start site for the lux mRNA was 26 bases before the initiation codon of the first gene, luxC. The promoter region contained a typical -10 but not a recognizable -35 consensus sequence. By using S1 nuclease mapping the mRNA was found to be induced in a cell density- and arginine-dependent manner. The DNA downstream of the five known V. harveyi lux genes, luxCDABE, was sequenced and found to contain coding regions for two new genes, designated luxG and luxH, followed by a classical rho-independent termination signal for RNA polymerase. luxG codes for a protein of 233 amino acids with a molecular weight of 26,108, and luxH codes for a protein of 230 amino acids with a molecular weight of 25,326. The termination signal is active in vivo as demonstrated by 3' S1 nuclease mapping, confirming that the two genes are part of the V. harveyi lux operon. Comparison of the luxG amino acid sequence with coding regions immediately downstream from luxE in other luminescent bacteria has demonstrated that this gene may be a common component of the luminescent systems in different marine bacteria.

Mapping of the puh Messenger RNAs from Rhodospirillum rubrum: Evidence for tandem promoters

Abstract The mRNA transcripts of Rhodospirillum rubrum gene puh, coding for the H subunit of the photoreaction center, and of genes flanking puh were analyzed by blot hybridization. Open reading frame G115, upstream of structural gene puh, is transcribed as a 2.25-kilobase mRNA. Gene puh itself is transcribed as two mRNAs of 1118 and 1032 nucleotides. Mung bean nuclease protection analysis shows that the puh transcripts have different 5' termini within open reading frame G115 and a unique rho-independent termination signal within open reading frame I2372. The lifetimes of the puh messages, as determined by an oxygen blockade of transcription, were 10 and 12 min for the large and small puh mRNAs, respectively. An expression vector carrying a chloramphenicol acetyltransferase gene was used to select promoters in DNA stretches upstream of the startpoints of each of these transcripts. Chloramphenicol resistance was expressed in Escherichia coli, using as a promoter a 179-nucleotide stretch upstream of the small mRNA startpoint but not from a 124-nucleotide stretch upstream of the large mRNA startpoint. The promoter for the small mRNA, designated Ppuh2, is thought to encompass in its -10 and -35 regions a sigma 70-like RNA polymerase recognition sequence. The region upstream of the large message startpoint contains a sequence similar in its -12 and -24 regions to promoter sequences recognized by the sigma 60 RNA polymerase holoenzyme. This is designated as promoter Ppuh1.Ppuh1 is proposed to be strictly regulated by light intensity and by oxygen tension while Ppuh2 would be less sensitive to these parameters.

Genetic characterization and sequence analysis of the duplicated nifA/nifB gene region of Rhodobacter capsulatus.

A DNA region showing homology to Klebsiella pneumoniae nifA and nifB is duplicated in Rhodobacter capsulatus. The two copies of this region are called nifA/nifB copy I and nifA/nifB copy II. Deletion mutagenesis demonstrated that either of the two copies is sufficient for growth in nitrogen-free medium. In contrast, a double deletion mutant turned out to be deficient in nitrogen fixation. The complete nucleotide sequence of a 4838 bp fragment containing nifA/nifB copy I was determined. Two open reading frames coding for a 59,653 (NifA) and a 49,453 (NifB) dalton protein could be detected. Comparison of the amino acid sequences revealed that the R. capsulatus nifA and nifB gene products are more closely related to the NifA and NifB proteins of Rhizobium meliloti and Rhizobium leguminosarum than to those of K. pneumoniae. A rho-independent termination signal and a typical nif promoter region containing a putative NifA binding site and a consensus nif promoter are located within the region between the R. capsulatus nifA and nifB genes. The nifB sequence is followed by an open reading frame (ORF1) coding for a 27721 dalton protein in nifA/nifB copy I. DNA sequence analysis of nifA/nifB copy II showed that both copies differ in the DNA region downstream of nifB and in the noncoding sequence in front of nifA. All other regions compared, i.e. the 5' part of nifA, the intergenic region and the 3' part of nifB, are identical in both copies.

The kdsA gene coding for 3-deoxy-D-manno-octulosonic acid 8-phosphate synthetase is part of an operon in Escherichia coli.

The kdsA gene of Escherichia coli was sequenced. It consists of 284 codons and is the last gene of a larger transcription unit. The mRNA is terminated by a rho-independent termination signal with the potential to be active in both orientations. This region is followed by another termination signal which seems to be active in the opposite orientation. Upstream of the kdsA gene a second open reading frame (ORF) was found; both this and the kdsA gene are transcribed in the same mRNA molecule when coded from the chromosome. In a plasmid-carried insert transcription starts from a cryptic promoter within the ORF preceding the kdsA gene. This promoter is not active in the intact chromosome.

Structural and functional organization of the gpt gene region of Escherichia coli

The nucleotide sequence was determined for the Escherichia coli region containing the gpt gene, which encodes the enzyme xanthine-guanine phosphoribosyi transferase (XGPRT; EC 2.4.2.22). Restriction enzyme and sequence analyses have allowed us to locate precisely the gpt gene (16.9-kDal XGPRT) with respect to other genes in this region, notably phoE. Genes gpt and phoE are pointing towards each other and are separated by about 1840 bp. Available sequence data and protein analyses [Overbeeke et al. J. Mol. Biol. 163 (1983) 513–522, and this paper] indicate the presence, between gpt and phoE, of two additional genes. These genes are oriented the same way as gpt and code for proteins of 49 and 15.7 kDal, respectively. By in vitro transcription with E. coli RNA polymerase and nuclease S l analysis, we have identified a promoter upstream of gpt. The short intercistronic region between gpt and the 49-kDal protein gene contains a rho-independent termination signal that closely precedes and partially overlaps another promoter. It appears from these data that gpt transcription is essentially monocistronic, giving rise to RNA of approx. 555 nucleotides, whereas the 49-kDal and 15.7-kDal protein genes are transcribed from their own promoter.

Initiation and termination signals for transcription in bacteriophage M13.

Transcription of the infrequently expressed phage M13 genome domain, comprising genes III, VI, I and IV, has been studied in detail by hybridization and S1-nuclease mapping studies. The contiguous genes III and VI are transcribed via an 1800 nucleotide-long RNA molecule that is initiated at a promoter which overlaps with the Rho-independent termination signal between genes III and VIII. Its synthesis is terminated at a Rho-dependent terminator in the proximal part of gene I. Transcription of gene I is not mediated by an independent promoter but most probably by read-through of RNA-polymerase through this terminator. Transcription of gene IV is accomplished by synthesis of four distinct RNAs of about 1500 to 1680 nucleotides long which are initiated at a promoter located immediately in front of gene IV. Termination of these transcripts is generated at least four different sites located in tandem within the intergenic region between genes IV and II.

Primary structure of the tolC gene that codes for an outer membrane protein of Escherichia coli K12.

We present the nucleotide sequence of the tolC gene of Escherichia coli K12, and the amino acid sequence of the TolC protein (an outer membrane protein) as deduced from it. The mature TolC protein comprises 467 amino acid residues, and, as previously reported (1), a signal sequence of 22 amino acid residues is attached to the N-terminus. The C-terminus of the gene is followed by a stem-loop structure (8 base pair stem, 4 base loop) which may be a rho-independent termination signal. The codon usage of the gene is nonrandom; the major isoaccepting species of tRNA are preferentially utilised, or, among synonomous codons recognized by the same tRNA, those codons are used which can interact better with the anticodon (2,3). In contrast to the codon usage for other outer membrane proteins of E. coli (4) the rare arginine codons AGA and AGG are used once and twice respectively.

Nut holding tension-grip nut drivers

The genes for translational components frequently are located together on the Escherichia coli genome. We have reported previously that the gene for a serine tRNA lies directly downstream from infA, the gene encoding initiation factor IF1. Here we characterize this tRNA gene, named serW. The serW gene expresses a minor form of serine tRNAGGA which recognizes the most frequently used serine codons, UCC and UCU. Two promoters were identified by S1 nuclease mapping: P1, which lies about 72 bp upstream from the structural gene; and P2, which lies about 35 bp upstream. Expression from P1 and P2 is comparable under conditions of rapid growth. The P2 promoter is followed by a GC-rich element characteristic of promoters regulated by ppGpp. A putative hairpin structure followed by a stretch of U residues about 25 nucleotides following the mature tRNA sequence resembles a rho-independent termination signal. The upstream gene, infA, is followed by a transcriptional terminator, but S1 mapping shows considerable readthrough. Thus serW expression appears to rely both on its own promoters and on promoters further upstream. The downstream gene, encoding an unidentified protein of about 100 kDa, is expressed in the opposite orientation and also is followed by a termination signal. Therefore serW is expressed both as a monocistronic gene and in combination with infA.