Putative Ribosome-binding Site Research Articles

Molecular cloning, characterization, and complete nucleotide sequence of the gene for pneumolysin, the sulfhydryl-activated toxin of Streptococcus pneumoniae.

A recombinant lambda bacteriophage has been isolated that carries DNA from Streptococcus pneumoniae and expresses a potent hemolysin that has been shown to be pneumolysin, the sulfhydryl-activated toxin of the pneumococcus. Hemolytic activity is inhibited by cholesterol and neutralized by serum against streptolysin O. The cloned gene expresses two polypeptides (Mrs, 56,000 and 53,000) in an Escherichia coli in vitro transcription-translation system, and both are precipitated by the addition of anti-alveolysin serum and anti-streptolysin O serum in the presence of Staphylococcus aureus cells. Expression of pneumolysin occurs when the gene is cloned in both possible orientations in pUC8. The DNA sequence of a 5-kilobase ClaI fragment that carries the pneumolysin gene has been determined. An open reading frame was identified that encodes a polypeptide of 471 amino acids that is hydrophobic in character and has an N-terminal amino acid sequence which is identical to that deduced from amino acid sequencing of the purified protein. The predicted amino acid sequence of the polypeptide reveals a single cysteine residue located 44 residues from the C terminus. Putative promoter and ribosome binding sites have been identified 5' to the pneumolysin coding sequence.

Cloning, sequencing, in vivo promoter mapping, and expression in Escherichia coli of the gene for the HhaI methyltransferase.

A 1476-base pair DNA fragment from Haemophilus haemolyticus containing the HhaI methyltransferase gene was isolated from a cell library and cloned into pBR322. The nucleotide sequence of this fragment was determined. The structural gene is 981 nucleotides in length coding for a protein of 327 amino acids (Mr 37,000). The translational start signal (ATG) is preceded by the putative ribosome-binding site (TAAG). Recombinant plasmids containing the 1476-basepair fragment are completely methylated when isolated from Escherichia coli, as judged by their insusceptibility to the HhaI restriction endonuclease. However, the presence of an active HhaI methylase gene in certain E. coli strains results in a very poor yield of transformants and/or in vivo-originated deletions due to the Rg1 functions of these hosts. The in vivo transcription initiation sites have been identified by S1 protection and primer-extension experiments using specific probes with total RNA prepared from E. coli cells (HB101 or RR1) which tolerate the expression of MHhaI.

Nucleotide sequence analysis of the structural genes for Shiga-like toxin I encoded by bacteriophage 933J from Escherichia coli

A nucleotide sequence analysis was performed on the Shiga-like toxin I genes previously cloned from the Escherichia coli bacteriophage 933J. Two structural genes designated slt-I A and slt-I B were found to be oriented on a single transcriptional unit with slt-I A preceding slt-I B. A 12 base pair gap separated the in-phase open reading frames of slt-I A and slt-I B. Putative ribosome binding sites were identified 5′ to both the slt-I A and slt-I B genes. Translation of the SLT-I nucleotide sequence revealed that both the A and B subunits were synthesized with signal peptides. The molecular weight calculated for the mature A subunit was 32211 while the molecular weight of the mature B subunit was 7690.

Cloning of DNA fragments containing Streptomyces promoter activity.

The thiostrepton-resistance gene is expressed in Streptomyces jumonjinensis [16]-8 SANK 61185 carrying the plasmid pIJ702 but not the tyrosinase gene. We have isolated DNA fragments from various streptomycetes that restore expression of the tyrosinase gene on pIJ702 in this organism. The nucleotide sequences of two of these DNA fragments show that they contain putative ribosome binding sites and- 10 regions of possible promoters.

Identification and characterization of the genes encoding three structural proteins of the Sulfolobus virus-like particle SSV1

Three structural proteins, VP1, VP2 and VP3, of the virus-like particle SSV1 of the thermoacidophilic archaebacterium Sulfolobus sp. strain B12 were purified. VP1 and VP3 are very hydrophobic and show a high degree of homology. They consist of 73 and 92 amino acid residues, respectively. The third protein, VP2, is extremely basic containing 29 basic amino acids but only 4 acidic ones in a total of 74 amino acid residues. The genes encoding these three proteins were mapped within the genome by comparison of N-terminal amino acid sequences with the SSV1 DNA sequence. The three genes are closely linked in the order VP1-VP3-VP2 and the coding strand is the same in all three genes. Ten nucleotides separate the stop codon for VP1 from the initiation codon for VP3 and one nucleotide separates the genes encoding VP3 and VP2. Duplicate putative ribosome binding sites are found upstream of the initiation codons for VP2 and VP3. The major coat protein VP1 does not start with a methionine residue and appears to be the result of proteolytic cleavage of a precursor molecule. Transcription of the region encoding VP1, VP2 and VP3 results in the formation of two mRNAs of 0.5 kb and 1.0 kb, the shorter one only encoding VP1, the longer one spanning all three genes. A 61 bp sequence encoding part of VP1 is exactly repeated within the gene for VP3 and these identical sequences are translated into stretches of identical amino acids in the two proteins. A function of this repeated DNA sequence beyond its coding properties is very likely.

The central part of the cyanelle rDNA unit of Cyanophora paradoxa: Sequence comparison with chloroplasts and cyanobacteria

The 287-bp spacer and the flanking 3'-end of the 16S- and 5'-end of the 23S-rRNA genes of the cyanelles from Cyanophora paradoxa have been sequenced and compared with the corresponding regions of cyanobacteria and chloroplasts. The spacer contains the uninterrupted genes for tRNA(ile) and tRNA(ala). All coding regions show high homology to their prokaryotic counterparts. At the 3'-end of the 16S-rDNA a CCTCCTTT sequence has been identified which is complementary to putative ribosome binding sites observed immediately upstream of the coding region of cyanelle protein genes.

Blogenesis of mitochondria: a mutation in the 5‘-untranslated region of yease mitochondrialolilmRNA leading to impairment in translation of subunit 9 of the mitochondrial ATPase Complex

A temperature-conditional mit- mutant of Saccharomyces cerevisiae has been characterized; the mutant strain h45 cannot grow at 36 degrees C on nonfermentable substrates yet appears to be normal at 28 degrees C. The mutation in strain h45 maps genetically to the oli1 region of the mitochondrial DNA (mtDNA) genome, and prevents the synthesis at 36 degrees C of the oli1 gene product, subunit 9 of the mitochondrial ATPase complex. Since the level of oli1 mRNA in mutant h45 is close to normal at 36 degrees C, it is concluded that there is a specific block in translation of this mRNA at the non-permissive temperature. DNA sequence analysis of mtDNA from strain h45 reveals an additional T residue inserted 88 bp upstream of the oli1 coding region, in the A,T-rich sequence that is transcribed into the 5'-untranslated region of the oli1 mRNA. Sequence data on two revertants show that one returns to wild-type parental (J69-1B) mtDNA sequence, whilst the other contains an inserted A residue adjacent to the T inserted in the original h45 mutant. The results are discussed in terms of the stability of folds in RNA upstream of putative ribosome-binding sites in mitochondrial mRNA, and the potential action of nuclear-coded proteins that might be activators of the translation of specific mitochondrial mRNAs in yeast mitochondria.

Primary structure of CTP:CMP-3-deoxy-D-manno-octulosonate cytidylyltransferase (CMP-KDO synthetase) from Escherichia coli.

The gene coding for CTP:CMP-3-deoxy-D-mannooctulosonate cytidylyltransferase (CMP-KDO synthetase), kds B, was previously cloned on a 9-kilobase Pst insert of Escherichia coli DNA into pBR 322 (Goldman, R. C., and Kohlbrenner, W. E. (1985) J. Bacteriol. 163, 256-261). Using a transposon mutagenesis approach we have now located kds B on this insert, which facilitated the isolation and sequencing of a 1.3-kilobase segment of DNA containing kds B and putative RNA polymerase and ribosome binding sites. The primary structure of CMP-KDO synthetase predicted by this nucleotide sequence was verified by amino acid composition and sequence analysis of purified CMP-KDO synthetase and cleavage fragments. Our results show that kds B consists of a 744-base open reading frame coding for a 248-amino acid peptide. The molecular weight of CMP-KDO synthetase calculated from the translated sequence is 27,486, taking into account the loss of the N-terminal methionine. These data define the transcriptional unit of kds B and its translation product in molecular terms, information prerequisite to our understanding of both the mechanism of CMP-KDO formation and the regulation of the KDO metabolic pathway in Gram-negative bacteria.

Characterization of the developmentally regulated Bacillus subtilis glucose dehydrogenase gene.

The DNA sequence of the structural gene for glucose dehydrogenase (EC 1.1.1.47) of Bacillus subtilis was determined and comprises 780 base pairs. The subunit molecular weight of glucose dehydrogenase as deduced from the nucleotide sequence is 28,196, which agrees well with the subunit molecular weight of 31,500 as determined from sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The sequence of the 49 amino acids at the NH2 terminus of glucose dehydrogenase purified from sporulating B. subtilis cells matched the amino acid sequence derived from the DNA sequence. Glucose dehydrogenase was purified from an Escherichia coli strain harboring pEF1, a plasmid that contains the B. subtilis gene encoding glucose dehydrogenase. This enzyme has the identical amino acid sequence at the NH2 terminus as the B. subtilis enzyme. A putative ribosome-binding site, 5'-AGGAGG-3', which is complementary to the 3' end of the 16S rRNA of B. subtilis, was found 6 base pairs preceding the translational start codon of the structural gene of glucose dehydrogenase. No known promoterlike DNA sequences that are recognized by B. subtilis RNA polymerases were present immediately preceding the translational start site of the glucose dehydrogenase structural gene. The glucose dehydrogenase gene was found to be under sporulation control at the trancriptional level. A transcript of 1.6 kilobases hybridized to a DNA fragment within the structural gene of glucose dehydrogenase. This transcript was synthesized 3 h after the cessation of vegetative growth concomitant to the appearance of glucose dehydrogenase.

Location and nucleotide sequence of the gene for cytochrome b-559 in wheat chloroplast DNA

The gene for the apoprotein of cytochrome b-559 has been located in wheat chloroplast DNA by coupled transcription-translation of fragments of chloroplast DNA in a cell-free system from Escherichia coli and by nucleotide sequence analysis. The gene is located 1.5 kbp downstream from the gene for cytochrome f and is transcribed in the opposite direction. Nucleotide sequence determination revealed an open reading frame coding for a hydrophobic protein of 83 amino acid residues (Mr 9434). The deduced amino acid sequence of the first 27 codons corresponds exactly to the determined N-terminal amino acid sequence of spinach cytochrome b-559. Another open reading frame of 39 codons is located 10 bp downstream from the gene for cytochrome b-559. A putative ribosome-binding site, AGGAGG, for this open reading frame overlaps the translation stop codon, UAG, of the cytochrome b-559 gene. The deduced amino acid sequence from this open reading frame shows similarities to the N-terminal sequence of cytochrome b-559. Both sequences contain a histidine residue located in a putative membrane-spanning region of the polypeptide.

Cytochrome b-559 genes from Oenothera hookeri and Nicotiana tabacum show a remarkably high degree of conservation as compared to spinach

Recent data suggest that cytochrome b-559, an intrinsic membrane protein of the oxygen-evolving photosystem II in chloroplasts, is a heme cross-linked heteromeric polypeptide unit (Herrmann et al. 1984, FEBS Lett 176:239-244). The genes for this cytochrome, designated psbE and psbF, have been located on the chloroplast chromosome of Oenothera hookeri and Nicotiana tabacum by hybridization with fragments of the corresponding spinach genes, and characterized. In both cases, the nucleotide sequence discloses 2 uninterrupted reading frames of 83 and 39 codons separated by a few nucleotides, as in spinach. The amber translation stop codon of psbE overlaps the putative ribosome-binding site for psbF in all cases. The predicted molecular weights of the proteins are 9.2 and 4.3 kd, respectively. In each of the three plant species, and the direction of transcription is opposite to that of the gene for cytochrome f which is located distal to the b-559 genes. Comparison of the deduced amino acid sequences with those from the corresponding spinach genes shows 97% homology. The ubiquitous presence and remarkably high degree of structural and functional conservation of this gene cluster supports the hypothesis of a heteromeric assembly and an important functional role for cytochrome b-559.

Sequencing and heterologous expression of the gene encoding penicillin V amidase from Bacillus sphaericus

The Bacillus sphaericusgene encoding penicillin V amidase, which catalyzes the hydrolysis of penicillin V, has been characterized.The entire nucleotide sequence of the coding region, as well as 5'- and 3'-flanking regions, was determined using an improved sequencing strategy.The deduced amino acid sequence suggests a protein consisting of 338 residues with an M rof 37500.The ATG initiator codon is preceded by a putative ribosome-binding site, typical for genes of Gram-positive origin.High expression of the gene was obtained in Escherichia coliusing an inducible promoter,showing that the gene product is stable in this heterologous host.

Nucleotide sequence of the right early region of Bacillus subtilis phage PZA completes the 19366-bp sequence of PZA genome. Comparison with the homologous sequence of phage φ29

We have sequenced the rightmost 2079 bp of the Bacillus subtilis phage PZA genome. This region encompasses the right early region. We compared it with the homologous region of phage φ29. Six open reading frames (ORFs) were found in this region of PZA and one of them was assigned to gene 17. Analysis of putative ribosome-binding sites and comparison with φ29 ORFs indicate that at least some of the remaining ORFs could encode proteins. Corresponding genes were not identified so far by genetic methods. Promoter candidates in the right early region of PZA were found and compared to φ29 promoters. The sequenced region together with previously determined sequences [Pačes et al., Gene 38 (1985) 45–56 and 44 (1986) 107–114] completes the entire 19366-bp sequence of phage PZA genome.

The nucleotide sequence and some properties of the neutral protease gene of Bacillus amyloliquefaciens

A 1.9 kb fragment possessing a gene for extracellular neutral protease of Bacillus amyloliquefaciens was cloned in pNP150. The nucleotide sequence of the fragment revealed putative regions which were considered as a promoter, a ribosome binding site and a transcription terminator as well as a protein coding region. The sequences of −35 and −10 regions of the putative promoter, TTGCAG and TATTAT, resembled the consensus sequences for the promoter recognized by the σ 55 RNA polymerase of Bacillus subtilis. The putative ribosome binding site, whose sequence was AAAGGGGG, was completely complementary to the binding sequence of B. subtilis 16S rRNA except for one base. The protein coding region which started at an initiation codon of GTG contained a sequence corresponding to a 221-residue-long stretch of amino acids preceding the N-terminal of the mature enzyme. This suggested that the neutral protease might have a pre-pro structure involved in an unknown function during secretion. The N-terminal amino acid sequence of the mature enzyme secreted by B. subtilis (pNP150) was the same as that of the authentic enzyme produced by B. amyloliquefaciens. This indicated that the pre-pro structure of B. amyloliquefaciens neutral protease synthesized in B. subtilis was exactly recognized and processed during secretion.

Phosphate-specific transport system of Escherichia coli: nucleotide sequence and gene-polypeptide relationships.

The DNA nucleotide sequence of four genes for the phosphate-specific transport system of Escherichia coli is reported. Along with the DNA sequence for the phoS gene reported previously (Surin et al., J. Bacteriol. 157:772-778, 1984; Magota et al., J. Bacteriol. 157:909-917, 1984), this study completes the nucleotide sequence of the phosphate-specific transport region. The complete sequence (including phoS) contains five open reading frames oriented in the same direction, each preceded by a putative ribosome-binding site near the presumed translation initiation codon ATG. The complete sequence is transcribed counterclockwise, in the order phoS pstC pstA pstB phoU. Genetic complementation shows that of the four open reading frames in the new sequence, three correspond to known mutant alleles; the fourth, which was designated pstC, has not been described before and could not be related to any known mutant allele. We have confirmed that pstA was allelic to phoT32. The pstC, pstB, and phoU gene products were identified as peripheral membrane proteins. The pstA gene product appears to be an integral membrane protein.

Cloning, sequencing and transcription of an inactivated copy of Bacillus amyloliquefaciens extracellular ribonuclease (barnase)

The gene for Bacillus amyloliquefaciens extracellular RNase (barnase) has been cloned in an inactive form in Escherichia coli following insertional mutagenesis by transposon Tn 977. The nucleotide (nt) sequence of the gene was determined and the deduced amino acid (aa) sequence found to correspond almost precisely to the previously determined sequence. An open reading frame (ORF) of 72 codons precedes the mature sequence. The probable translation start site is 46 or 47 codons before the N-terminal alanine of the mature protein, 11 (or 14) bp from a putative ribosome-binding site (RBS). Within this leader sequence is a hydrophobic 15 aa core preceded by three basic residues which is characteristic of a secretory signal sequence. A pro-barnase protein with four extra aa at the N-terminus has been detected extracellularly indicating that the signal peptidase-cutting site lies before the mature protein. An inverted repeat that may act as a transcription terminator was found at the 3′ end of the gene. The gene is maintained in E. coli with a short inverted repeat from the termini of Tn 917 inserted into the coding sequence. Northern blot analysis of RNA from B. amyloliquefaciens shows an approx. 780-nt transcript produced during exponential and stationary growth phases. The inactive cloned gene produces an approx. 480-nt transcript in E. coli and two transcripts of approx. 480 and 780 nt in Bacillus subtilis.

The primary structure of phosphoenolpyruvate carboxylase of Escherichia coli. Nucleotide sequence of the ppc gene and deduced amino acid sequence.

The nucleotide sequence of the ppc gene, the structural gene for phosphoenolpyruvate carboxylase [EC 4.1.1.31], of Escherichia coli K-12 was determined. The gene codes for a polypeptide comprising 883 amino acid residues with a calculated molecular weight of 99,061. The amino acid sequence deduced from the nucleotide sequence was entirely consistent with the protein chemical data obtained with the purified enzyme, including the NH2- and COOH-terminal sequences and amino acid composition. The coding region is preceded by two putative ribosome binding sites, and is followed closely by a good representative of rho-independent terminator. The codon usage in the ppc gene suggests a moderate expression of the gene. The secondary structure of the enzyme was predicted from the deduced amino acid sequence.

Nucleotide sequence of DNA controlling expression of genes for maltosaccharide utilization in Streptococcus pneumoniae

An analysis of previous data indicated that four structural genes concerned with maltosaccharide utilization in Streptococcus pneumoniae are organized in two operons that are transcribed in opposite directions from a central control region. This region contains two strong promoters subject to repression by a regulatory gene product in the absence of maltose. The nucleotide sequence of the 554-bp control region DNA and adjacent portions of the malX and malM structural genes was determined. Unique reading frames and initiation codons allowed identification of the oppositely oriented structural genes. Putative ribosome binding sites and −10 and −35 RNA-polymerase-binding sites, as well as AT-rich regions farther upstream, were observed proximal to both the X and M genes. The similarity of these sequences to sites found in Escherichia coli and Bacillus subtilis indicated the conservation of control signals in bacteria, both Gram-negative and Gram-positive. A pair of 17-bp hyphenated repeat sequences in the control region may represent repressor binding sites. Two down promoter mutations, V11 and 69, were shown to be deletions in the control region. The V11 mutation, which affected only the MP operon, deleted the promoter adjacent to the M gene. Mutation 69, which reduced both X and M gene functions, deleted the entire segment between the promoters so that they now overlap at their −35 binding sites. As a consequence of this deletion, the AT-rich regions proximal to the promoters were lost. This suggests that the AT-rich regions are important for promoter strength.

The nucleotide sequence of the tobacco chloroplast gene for the large subunit of ribulose-l,5-bisphosphate carboxylase/oxygenase

The gene for the large subunit (LS) of ribulose-l,5-bisphosphate carboxylase/oxygenase (RuBPCase/ Oase) from tobacco has been cloned in pBR322 and sequenced. The coding region contains 1431 bp (477 codons). The deduced arnino acid sequence of tobacco LS protein shows 90% homology with those of maize and spinach LS. The positions in the gene corresponding to the 5' and the 3' ends of tobacco LS mRNA have been located on the DNA sequence by the S1 nuclease mapping procedure. The LS gene promoter sequence has homology with Escherichia coli promoter sequences; its terminator sequence is capable of forming a stem-and-loop structure. A sequence GGAGG, which is complementary to a sequence near the 3' end of tobacco chloroplast 16S rRNA and a putative ribosome binding site, occurs 6–10 bp upstream from the initiation codon.

Sequence of the gene coding for the β-subunit of dinitrogenase from the blue-green alga Anabaena

The nitrogen fixation nif K gene of the blue-green alga Anabaena, which codes for the beta-subunit of dinitrogenase, has been subjected to sequence analysis. The nif K protein is predicted to be 512 amino acids long, to have a M(r) or 57,583, and to contain six cysteine residues. Three of these cysteines are within peptides homologous to FeS cluster-binding cysteinyl peptides from ferredoxins and from a high potential iron protein and, thus, may be ligands to which FeS clusters bind in dinitrogenase. The sequences surrounding the cysteine residues are 70% homologous to the corresponding cysteinyl tryptic peptides of the Azotobacter vinelandii dinitrogenase, although the positions of the cysteine residues are not always conserved between the two proteins. A 15-amino acid coding sequence precedes nif K on its transcript. Amino acid codon usage is highly asymmetric and parallels that found for the Anabaena dinitrogenase reductase gene (nif H). Putative promoter and ribosome binding site sequences were identified for nif K. These regulatory sequences are homologous to sequences preceding nif D; nif D codes for the alpha-subunit of dinitrogenase but is separated from nif K on the chromosome by 11,000 nucleotides. The nif K promoter also is virtually identical to a promoter-like sequence that immediately precedes the start of the transcript for the large subunit of ribulosebisphosphate carboxylase from maize chloroplasts. This homology appears to support the theory that chloroplasts evolved from blue-green algae.