Potential Ribosome Binding Research Articles

The alpha 2 cDNA sequence of human haptoglobin carries a bacterial promoter functional in vivo.

Various constructions of human haptoglobin (Hp) cDNA coding either for the complete alpha 2FS beta precursor protein or only for the beta subunit have been placed under the control of the lambda PR promoter in the bacterial expression vector pCQV2 (Queen, 1983). In addition to the expected 45,000 dalton polypeptide synthesized after induction of the PR promoter, the complete alpha 2FS beta constructions constitutively express a smaller polypeptide of approximately 30,000 dalton corresponding to a truncated Hp protein. Computer analysis of the HpcDNA revealed the presence of two strong potential bacterial promoters (alpha 2 PF and alpha 2 PS) located in the duplicated alpha 2FS sequence. Both Hp promoter signals are followed by potential mRNA start sites and ribosome binding sites at a compatible distance from initiation codons. In addition, the Hp alpha 2 cDNA sequence, when fused upstream to the cDNA coding for alpha 1-antitrypsin, constitutively promotes in vivo the efficient expression of an hybrid protein specifically recognized by antibodies raised against alpha 1-antitrypsin or haptoglobin.

Primary structure and expression of a gene homologous to nifH (nitrogenase Fe protein) from the archaebacterium Methanococcus voltae

In Methanococcus voltae, a 3.0 kbp HindIII fragment carrying homology to nifH was recently cloned. In Escherichia coli maxicells, the fragment directed the synthesis of a 30 K polypeptide encoded by the region homologous to nifH. Plasmids carrying the fragment did not complement Klebsiella pneumoniae nifH mutants and did not inhibit the nitrogen fixation of a Nif+ strain. The complete nucleotide sequence of the nifH homologous region was determined. It contained an open reading frame (ORFnifH) of 834 bp encoding 278 amino acid residues (mol. wt. 30,362). The ORFnifH was surrounded by regions of very high A+T content as observed with other mc. voltae genes. The region upstream from ORFnifH contained potential prokaryotic-like promoters and a potential ribosome binding site located 5 bp preceding the translation initiation codon. Using a translational fusion to lacZ of a DNA fragment carrying the putative promoter region and the 5′ end of ORFnifH, it was shown in E. coli that (i) a promoter activity was effectively carried by the cloned fragment and (ii) this activity was not significantly modified by the presence of nifA or ntrC products provided by multicopy plasmids. Though the codon usage was characteristic of Mc. voltae, ORFnifH was very similar to eubacterial nifH genes, in particular the position of the cysteine residues was highly conserved. These data confirmed the high conservation of nifH sequences. SAB values (binary matching coefficients) of 0.5 were found with eubacterial nifH genes at the nucleotide or amino acid level suggesting that the mc. voltae ORFnifH sequence was distantly related to eubacterial nifH sequences.

Sequence of two genes in pea chloroplast DNA coding for 84 and 82 kD polypeptides of the photosystem I complex

The genes encoding the two P700 chlorophyll a-apoproteins of the photosystem I complex were localized on the pea (Pisum sativum) chloroplast genome. The nucleotide sequence of the genes and the flanking regions has been determined. The genes are separated by 25 bp and are probably cotranscribed. The 5' terminal gene (psaA1) codes for a 761-residue protein (MW 84.1 kD) and the 3' terminal gene (psaA2) for a 734-residue protein (MW 82.4 kD). Both proteins are highly hydrophobic and contain eleven putative membrane-spanning domains. The homology to the corresponding polypeptides from maize are 89% and 95% for psaA1 and psaA2, respectively. A putative promoter has been identified for the psaA1 gene, and potential ribosome binding sites are present before both genes.

Nucleotide sequence of a major class-III phage-T3 RNA-polymerase promoter located at 98.0% of phage-T3 genetic map

The entire nucleotide sequence of a 409-bp HincII fragment, located within the MboI-E fragment on bacteriophage T3 DNA and containing a major class-III T3 RNA polymerase promoter positioned at 98% on the standard T3 genetic map, has been determined. Alignment of this class-III promoter with previously determined T3 RNA polymerase promoters, with start points of transcription (+1) in register, indicates high degree of sequence conservation between position −16 to +6 among all T3 RNA polymerase promoters. The conserved portion of the (−) strand sequence is 5'-A- A T-T- T A-A-C-C-C-T-C-A-C-T-A-A-A-G-G-G-A---3'. This fragment also contains an open reading frame (ORF) with a translational start codon located at position +146 which is preceded by a potential ribosome binding site (RBS). There is more than 70% amino acidsequence homology between the deduced sequences of the -NH 2 terminal region of this putative T3 phage protein and the corresponding protein coded by bacteriophage T7 (protein of T7 gene 19.5).

Nucleotide sequence of the clustered genes for the 44 kd chlorophyll a apoprotein and the ?32 kd?-like protein of the photosystem II reaction center in the spinach plastid chromosome

A 2,900 base pair DNA segment of the spinach plastid chromosome which encodes the genes for the 44 kd chlorophyll a apoprotein and a "32 kd"-like protein of the photosystem II reaction center has been subjected to sequence and Northern blot analysis. The genes are located almost centrally in the large single-copy segment of the chromosome adjacent to the two genes for the P700 chlorophyll a apoproteins of the photosystem I reaction center. The DNA sequence reveals two uninterrupted protein-coding regions of 473 (44 kd chlorophyll a apoprotein) and 353 triplets ("32 kd"-like protein). The latter gene is strikingly similar to the gene for the herbicide-binding "32 kd" protein which maps some 30 kbp distant on the plastid chromosome. The two genes overlap by 50 base pairs but are read in different phases. They may be contranscribed and the RNA modified to give several discrete species ranging in size from 1.6 to 4.6 kb. A presumptive promoter site was only identified for the "32 kd"-like protein, while potential ribosome binding and transcription termination sites are found preceding and following both genes, respectively. The polypeptides possess a high content of hydrophobic amino acids, most of which appear to be clustered in transmembrane spans. The molecular weights of 51,785 (44 kd chlorophyll a apoprotein) and 39,465 ("32 kd"-like protein) derived from the deduced amino acid sequences are higher than the experimentally determined protein sizes. Amino acid codon usage for both genes is highly selective. Comparison of the chlorophyll a apoproteins of spinach reveals regions of sequence homology.

The apocytochrome b gene in maize mitochondria does not contain introns and is preceded by a potential ribosome binding site

The apocytochrome b (COB) gene has been isolated from maize (Zea mays L.) mitochondrial DNA. Sequence analysis reveals that the coding region of the gene is 1164 bp long and, in contrast with the homologous gene from yeast, does not contain introns or TGA (Trp) codons. The predicted polypeptide encoded by the gene has a mol. wt. of 42 868 daltons, and shows 48% amino acid sequence homology with the corresponding yeast and mammalian polypeptides. Hydropathic profiles of the polypeptide indicate the presence of nine, membrane spanning hydrophobic domains suggesting that it is organised in the inner mitochondrial membrane in a similar fashion to that proposed for apocytochrome b in other organisms. The COB gene is preceded by a sequence 5' -AGTTGTCA-3' which may act as a ribosome binding site in the mRNA since: (i) it shows 67.5% complementarity with an octanucleotide at the 3' end of the maize mitochondrial 18S rRNA, located in a position homologous to that of the Escherichia coli Shine and Dalgarno sequence, and (ii) a similar sequence precedes several other plant mitochondrial genes at a distance of 15-20 nucleotides from the ATG initiation codon. RNA transcript analysis shows that the gene is transcribed in a complex manner with the presumed mature mRNA ( 2.25 kb) probably being derived by sequential processing from a larger primary transcript.

Formation of termination-resistant transcription complex at phage lambda nut locus: effects of altered translation and a ribosomal mutation.

Transcription antitermination by lambda N gene product is affected in a mutant Escherichia coli with altered ribosomal protein S10, caused by the nusE71 mutation. To study the role of translation in antitermination, we have fused the phage nutR locus, the site of action of N, with the lac regulatory region. We have monitored N action by measuring galactokinase, whose synthesis depends on suppression of terminators located between nutR and the galK cistron. We show that a deletion removing potential ribosome binding signals and AUG codons from the upstream region of nut site does not affect N action. Moreover, the lack of translation upstream of nutR does not overcome the antitermination defect caused by nusE mutation. When the upstream region is translated, however, N action is impaired if translation terminates 19 base pairs upstream of nutR . Termination of translation at further upstream sites, such as 23 or 97 base pairs upstream, does not interfere with N action. Our results suggest that the S10 ribosomal protein is required for N action without involving translation. These results also suggest that the nut site RNA itself plays an important role in the formation of a termination-resistant transcription complex.

DNA sequence and regulation of ermD, a macrolide-lincosamide-streptogramin B resistance element from Bacillus licheniformis.

The DNA sequence of ermD , a macrolide-lincosamide-streptogramin B (MLS) resistance determinant cloned from the chromosome of Bacillus licheniformis, has been determined. ermD encodes an erythromycin inducible protein of molecular weight 32,796. S1 nuclease mapping of the ermD promoter has revealed the presence of an approximately 354 base leader sequence on the ermD transcript. This leader contains a short open reading frame sufficient to encode a 14 amino acid peptide, which is preceded by a potential ribosomal binding site. The leader sequence has the potential to fold into several base paired structures, in some of which the ribosomal binding site for the ermD product would be sequestered. Deletion analysis demonstrated that the leader contains regulatory sequences. Removal of the ermD promoter and fusion to an upstream promoter did not interfere with induction, strongly suggestion that ermD regulation is posttranscriptional. Based on these features it appears likely that ermD is regulated by a translational attenuation mechanism, analogous to that suggested for ermC , a resistance element from Staphylococcus aureus ( Gryczan et al. 1980; Horinouchi and Weisblum 1980). Comparison of the ermD sequence and that of its product to two other sequenced MLS determinants reveals substantial phylogenetic relatedness, although the three genes are not homologous by the criterion of Southern blot hybridization.

Biogenesis of mitochondria: genetic and molecular analysis of the oli2 region of mitochondrial DNA in Saccharomyces cerevisiae

A genetic and molecular analysis of the oli2 region in mitochondrial DNA of Saccharomyces cerevisiae has been carried out. The oli2 gene codes for subunit 6 of the mitochondrial ATPase complex. We have isolated a series of 41 mit −, temperature sensitive and oligomycin resistant mutants containing mutations located in the oli2 region. In addition the sequence in wild type mtDNA of 2820 nucleotides covering the oli2 gene and its flanking regions has been determined. A library of petite mutants which have various deletion end points in the oli2 region has been used to generate marker rescue data that allow the assignment of each mit −, temperature sensitivity and oligomycin resistance mutation to one of seven uniquely ordered genetic groups. The following physical reference points allow the limits of the physical map positions of a number of the genetic groups to be established: DNA sequence data have been obtained for three of the petites so as to determine unambiguously their relevant deletion end points and the base substitution constituting the mtDNA sequence change in one oligomycin resistant mutant [oli2-23r] has been determined to lie at nucleotide +523 of the oli2 coding region. One group of mit − mutations (group N) are known to map within the aap1 gene that is found some 700 bases upstream of the oli2 gene, and which codes for subunit 8 of the mitochondrial ATPase complex. Mutations in the remaining six groups (A-F) map within (or very close to) the coding region of the oli2 gene. Various features of the DNA sequence in and around the oli2 gene are considered, including protein coding regions, sequence divergence in the A,T-rich spacer regions, properties of G,C-rich clusters, and potential ribosome binding sites. Consideration of the transcriptional map of this region of mtDNA suggests that an abundant 4,500 nucleotide transcript may represent the first defined example of a dicistronic messenger RNA in yeast mitochondria carrying distinct coding regions (aap1 and oli2) for two different proteins.

Biogenesis of Mitochondria: Genetic and molecular analysis of the oli2 region of mitochondrial DNA in Saccharomyces cerevisiae

A genetic and molecular analysis of the oli2 region in mitochondrial DNA of Saccharomyces cerevisiae has been carried out. The oli2 gene codes for subunit 6 of the mitochondrial ATPase complex. We have isolated a series of 41 mit (-), temperature sensitive and oligomycin resistant mutants containing mutations located in the oli2 region. In addition the sequence in wild type mtDNA of 2820 nucleotides covering the oli2 gene and its flanking regions has been determined. A library of petite mutants which have various deletion end points in the oli2 region has been used to generate marker rescue data that allow the assignment of each mit (-), temperature sensitivity and oligomycin resistance mutation to one of seven uniquely ordered genetic groups. The following physical reference points allow the limits of the physical map positions of a number of the genetic groups to be established: DNA sequence data have been obtained for three of the petites so as to determine unambiguously their relevant deletion end points and the base substitution constituting the mtDNA sequence change in one oligomycin resistant mutant [oli2-23r] has been determined to lie at nucleotide +523 of the oli2 coding region. One group of mit (-) mutations (group N) are known to map within the aap1 gene that is found some 700 bases upstream of the oli2 gene, and which codes for subunit 8 of the mitochondrial ATPase complex. Mutations in the remaining six groups (A-F) map within (or very close to) the coding region of the oli2 gene. Various features of the DNA sequence in and around the oli2 gene are considered, including protein coding regions, sequence divergence in the A,T-rich spacer regions, properties of G,C-rich clusters, and potential ribosome binding sites. Consideration of the transcriptional map of this region of mtDNA suggests that an abundant 4,500 nucleotide transcript may represent the first defined example of a dicistronic messenger RNA in yeast mitochondria carrying distinct coding regions (aap1 and oli2) for two different proteins.

Localization and nucleotide sequence of the gene for the membrane polypeptide D2 from pea chloroplast DNA

The gene for the membrane polypeptide D2 has been mapped on the pea (Pisum sativum) chloroplast genome. The nucleotide sequence of the gene and its flanking regions is presented. The only large open reading frame in the sequence codes for a protein of MW 39.5 kD. A potential ribosome binding site is located 6 nucleotides upstream from the initiation codon and there are two sets of putative promotor sequences in the 5' flanking region. The polypeptide has a high content of hydrophobic amino acids, predominatly grouped in clusters of 20 or more residues. The 3' end of the D2 gene is overlapped by 50 nucleotides of a second open reading frame (UORF I) which is at least 369 nucleotides long. Based on current data we suggest the D2 polypeptide to be a constituent of photosystem II (PSII).

Transcriptional and translational start sites for the Bacillus thuringiensis crystal protein gene.

The nucleotide sequence of the promoter region and part of the coding region of the crystal protein gene from Bacillus thuringiensis var. kurstaki HD-1-Dipel has been determined by analysis of a recombinant plasmid from Escherichia coli. The start points for transcription of the gene in B. thuringiensis and in the E. coli strain carrying the recombinant plasmid were located by S1 nuclease mapping. Two adjacent start sites were identified using RNAs synthesized during sporulation of B. thuringiensis: transcription was initiated from one site early in sporulation and from the other site in the middle of sporulation. A good correlation was found between the appearance of the crystal protein gene-specific RNA and the production of the protein, indicating that the gene is primarily under transcriptional control during sporulation. Parallel studies with the recombinant strain of E. coli revealed the presence of only a single species of gene-specific RNA, regardless of the growth phase of the cells; the crystal protein was produced at all stages of growth. The sequence for eight amino acids at the NH2 terminus of the crystal protein was determined and the corresponding coding sequence was located in the DNA sequence. A potential ribosome binding site of 11 nucleotides was found, located three nucleotides upstream from the initiator ATG codon. The deduced sequence for the first 333 amino acids of the crystal protein is presented.

Location and nucleotide sequence of the gene for the proton-translocating subunit of wheat chloroplast ATP synthase

The proton-translocating subunit of wheat chloroplast ATP synthase is encoded by a chloroplast gene that has been accurately mapped and whose nucleotide sequence has been determined. The predicted sequence of 81 amino acids has been confirmed in part by determination of the sequence of the first 40 amino acids from the NH(2) terminus of the protein, and it shows 100% homology with the known amino acid sequence of the spinach protein but no more than 35% homology with the amino acid sequences of bacterial and mitochondrial proteins. The gene shows no deviation from the "universal" genetic code and is not split. A potential ribosome binding site is located 12 nucleotides upstream from the initiation codon, but sequences homologous to prokaryotic promotors and transcription terminators are not apparent.

Nucleotide sequence of the major early region of bacteriophage φ29

The nucleotide sequence of the left end of bacteriophage φ29 DNA has been determined. Together with data reported earlier (Yoshikawa et al., 1981), this sequencing comprises the major early genetic region of this viral genome (5708 bp). Computer analysis of the DNA sequences revealed that there are up to fifteen open reading frames which could encode polypeptides containing more than thirty amino acids. The DNA sequence also revealed a number of potential regulatory signals, promoters and ribosome binding sites. The initiation and the termination of transcription and probable early gene products are discussed.

Nucleotide sequence and the encoded amino acids of human serum albumin mRNA.

The complete nucleotide sequence of human serum albumin mRNA has been determined from recombinant cDNA clones and from a primer-extended cDNA synthesis on the mRNA template. The sequence is composed of 2078 nucleotides, starting upstream from a potential ribosome binding site in the 5' untranslated region. It contains all the translated codons and extends into the poly(A) at the 3' terminus. Part of the translated sequence codes for a hydrophobic prepeptide, Met-Lys-Trp-Val-Thr-Phe-Ile-Ser-Leu-Leu-Phe-Leu-Phe-Ser-Ser-Ala-Tyr-Ser, followed by a basic propeptide, Arg-Gly-Val-Phe-Arg-Arg. These signal peptides are absent from mature normal serum albumin and, so far, have not been identified in their nascent state in humans. A remaining 1755 nucleotides of the translated mRNA sequence code for 585 amino acids, which are in agreement, with few exceptions, with the published amino acid sequence for human serum albumin. The mRNA sequence verifies and refines the repeating homology in the triple-domain structure of the serum albumin molecule.

Nucleotide sequences and mapping of novel heterogenous 5'-termini of adenovirus 2 early region 4 mRNA.

The major 5'-termini of human adenovirus type 2 early gene block 4 mRNA were sequenced. Poly(A+) polyribosomal RNA was isolated from Ad2 early infected cells, the 5'-terminal m7GPPP removed and the 5'-OH of the penultimate 2'-0-methylated nucleotide labeled with [gamma-32P]ATP using polynucleotide kinase. Ad2 E4 mRNA was purified by hybridization to the Ad2 EcoRI-C fragment and was digested with RNase T1. The resulting oligonucleotides were resolved by two dimensional paper electrophoresis-homochromatography. Four major and 3-4 minor 5'-terminal sequences were identified and characterized. The sequence of the 5'-terminal structures of the major four termini are: (1) m7GpppUmU(m)UUACACUGp, (2) m7GpppUmU(m)UACACUGp, (3) m7GpppUmU(m)ACACUGp, and (4) m7Gppp(m6)AmC(m)ACUGp. These major 5'-terminal sequences were aligned with nucleotide 325, 326, 327, and 329 from the righthand end of the known Ad2 DNA sequence (1) in the region mapped as the 5'-terminus of E4 mRNA by electron microscopy (2,3) and S1 nuclease-gel (4) mapping. Two potential ribosomal binding sites and an initiator codon were found at 40 to 65 nucleotides and about 80 nucleotides, respectively, from these heterogenous 5'-termini. Ad2 E4 major mRNA species appear to be unique since mRNA molecules initiate at a pyrimidine, perhaps by RNA polymerase stuttering, or they are products of an unusual type of RNA processing.

Nucleotide sequence and genetic organization of the polyoma late region: Features common to the polyoma early region and SV40

The nucleotide sequence of the late region of the polyoma genome has been determined. It consists of 2366 bp and encodes the virion capsid proteins VP1, VP2 and VP3. Extensive open reading frames identify the possible coding sequences of VP2 and VP3 toward the 5′ end of the late region, and of the major capsid protein VP1 toward the 3′ end of the late region. The 5′ end of the sequence encoding VP1 overlaps the 3′ VP2/VP3 region by 29 nucleotides and is in a different reading frame. The predicted amino acid sequences for all three known capsid proteins show extensive homology with the analogous capsid proteins of SV40 throughout most of their length. The VP2/VP3 amino acid homology between the two viruses is 34%, while the major capsid protein VP1 is much more highly conserved, showing 54% homology. These homologies together with the extent of open reading frames help to define the extent of the coding sequences. The VP2 initiator begins at position 269 and the coding region extends to the first termination codon beginning at 1226. The predicted size of VP2 is 35,007 daltons. A probable VP3 initiator is within the VP2 coding sequence at position 614 and is in the same frame as VP2. This coding sequence can also utilize the terminator at position 1226, and the predicted size of the VP3 translation product is 22,979 daltons. The VP1 coding region begins at position 1197 and continues in a frame different from that of VP2/ VP3 to a termination point at 2349. The molecular weight of VP1 is predicted to be 42,834 daltons. The 5′ untranslated region contains sequences that resemble a potential ribosomal binding site and a possible mRNA capping sequence similar to those found in other eucaryotic systems. There is also a sequence (5′-TCAAGTAAGTGA-3′) almost identical to one found in two regions containing potential splice sites in the early region of polyoma. The 5′ untranslated region does not show the extensive repeated sequences found in the similar region of SV40. The 3′ untranslated region contains the sequence 5′-AATAAA-3′, thought to represent a polyadenylation signal. As in the early region of polyoma, the extensive nucleotide and deduced amino acid homology with SV40 indicate a close evolutionary relationship between the two viruses, and help to identify regions of common and important structure-function relationships.