Nucleotide sequence of a cellulase gene of Bacillus subtilis

The nucleotide sequence of an endolytic cellulase gene of Bacillus subtilis was determined and compared with the NH2-terminal amino acid sequence of the purified enzyme. The mature protein appeared to be extended by a signal sequence of 36 amino acids. The putative AUG initiation codon was preceded by a sigma 43-type promoter of B. subtilis and an AAGGAGG sequence, typical of procaryotic ribosomal binding sites. Partial homology of amino acid sequences was found between B. subtilis cellulase and an alkalophilic Bacillus cellulase.     

Purification and characterization of the spoOA protein of Bacillus subtilis from an overproducing strain of Escherichia coli

The spoOA gene of Bacillus subtilis is essential for the earliest stage of sporulation. To purify and characterize the product of the spoOA gene, we constructed a fusion plasmid in which the spoOA coding region was placed under the control of the Ptac promoter. When expression of the spoOA gene was induced in Escherichia coli cells by derepression of Ptac, the SpoOA protein constituted 15% of total cellular protein. The SpoOA protein was purified to homogeneity from these cells. We found that the NH2-terminal amino acid sequence of the purified protein was essentially the same as that of the SpoOA protein (spoOA-cat protein) from B. subtilis, and that the NH2-terminal methionine of the SpoOA protein from E. coli was formylated presumably because of insufficient amounts of the deformylating enzyme. The T signal [Ganoza, M. C., Marliere, P., Kofoid, E. C. and Louis, B. G. (1985) Proc. Natl Acad. Sci. USA 82, 4587-4591], in addition to the Shine-Dalgarno signal to determine the initiation codon of the spoOA gene, is considered to function in E. coli as well as in B. subtilis. We also found that the purified SpoOA protein had a DNA-binding activity. It was preferentially bound to the 175-bp BclI fragment of phi 105 DNA, and was released in the presence of 0.3 M KCl.     

Nucleotide sequence of the membrane-bound aldehyde dehydrogenase gene from Acetobacter polyoxogenes

The nucleotide sequence of the membrane-bound aldehyde dehydrogenase (ALDH) gene from an industrial vinegar producer, Acetobacter polyoxogenes, was determined. Comparison of the sequence with the NH2-terminal amino acid sequence of the mature ALDH and determination of the actual translational initiation codon by means of in vitro manipulation of the upstream and proximal regions of the cloned gene showed that ALDH was primarily translated as a 773-amino-acid protein and that the 44-amino-acid sequence at the NH2-terminus, which probably serves as a signal peptide, was processed during maturation and localization in the membrane. When ALDH was expressed in a large quantity in Escherichia coli cells after the coding region had been placed downstream of the lac promoter, the ALDH protein, which still contained the signal peptide and had no ALDH activity, was localized in the membrane fraction.     

Role of ribosome recycling factor (RRF) in translational coupling

RNA phage GA coat and lysis protein expression are translationally coupled through an overlapping termination and initiation codon UAAUG. Essential for this coupling are the proximity of the termination codon of the upstream coat gene to the initiation codon of the lysis gene (either a <3 nucleotide separation or physical closeness through a possible hairpin structure) but not the Shine-Dalgarno sequence. This suggests that the ribosomes completing the coat gene translation are exclusively responsible for translation of the lysis gene. Inactivation of ribosome recycling factor (RRF), which normally releases ribosomes at the termination codon, did not influence the expression of the reporter gene fused to the lysis gene. This suggests the possibility that RRF may not release ribosomes from the junction UAAUG. However, RRF is essential for correct ribosomal recognition of the AUG codon as the initiation site for the lysis gene.     

Nucleotide sequence of a DNA fragment that contains the abi gene of the ColIb plasmid

A 1989-bp PstI DNA fragment from the ColIb plasmid, which contains the abi gene that is necessary for the abortive response to infections by bacteriophage BF23 or T5, was sequenced. A candidate open reading frame for the abi gene has been suggested on the basis of a Shine-Dalgarno sequence appropriately placed ahead of its ATG initiation codon, a promoter upstream from the Shine-Dalgarno sequence, and a location compatible with deletion mapping. The polypeptide that would be coded by this open reading frame is 89 amino acids long and strongly hydrophobic. A promoter that could serve this open reading frame was detected by exonuclease III "footprinting" using RNA polymerase from uninfected Escherichia coli as the DNA-binding protein.     

Structure of Escherichia coli dnaC. Identification of a cysteine residue possibly involved in association with dnaB protein

The nucleotide sequence of the Escherichia coli dnaC gene and the primary structure of the dnaC protein were determined. The NH2-terminal amino acid sequence of the dnaC protein matched that predicted from the nucleotide sequence of the 735-base pair coding region. The dnaC gene lacks characteristic promoter structures; neither the "Pribnow box" nor the "-35 sequence" was detected within 222 base pairs upstream from the initiator ATG codon. There is, however, a typical Shine-Dalgarno sequence 7-10 base pairs before the ATG codon. An upstream open reading frame, separated by just 2 base pairs from the coding region of dnaC, encodes the COOH-terminal half of the dnaT product (protein i; Masai, H., Bond, M. W., and Arai, K. (1986) Proc. Natl. Acad. Sci. U. S. A. 83, 1256-1260). The dnaC protein contains 245 amino acids with a calculated molecular weight of 27,894 consistent with the observed value (29,000). Similar to dnaG and dnaT, dnaC uses several minor codons; the significance of these minor codons to the low level expression of the protein product in E. coli cells remains to be determined. The in vitro site-directed mutagenesis method was employed to determine the functional region involved in interaction with dnaB protein. The first cysteine residue located in the NH2-terminal region of the dnaC protein (Cys69) was shown to be important for this activity. Overall sequence homology between dnaC protein and lambda P protein, functionally analogous to the dnaC protein in the lambda phage DNA replication, is not extensive. There are, however, several short stretches of homologous regions including the NH2-terminal eight amino acids and the Cys78 region of dnaC protein.     

Mutational analysis of an inherently defective translation initiation site.

In a reverse of many studies of translational initiation sites, we have explored the basis for the inactivity of an apparently defective initiation site. Gene VII of the filamentous phage f1 has a translational start site with highly unusual functional properties and a sequence dissimilar to a prokaryotic ribosome binding site. The VII site shows no activity in assays of independent initiation, even in a deletion series designed to remove potentially interfering RNA secondary structure. Activity from the VII site is only observed if the site is coupled to a source of translation immediately upstream, but its efficiency is low at a one-nucleotide spacing from the stop codon of the upstream cistron and extremely sensitive to the distance between the stop codon and the gene VII AUG. These and other atypical characteristics of coupling distinguish the VII site from most coupled initiation sites. To identify the pattern of nucleotide substitutions that give the VII site the capacity for independent initiation, a series of designed and random point mutations were introduced in the sequence. Improving the Shine-Dalgarno complementarity from GG to GGAG or GGAGG made activity detectable, but at only low levels. Random substitutions, each increasing activity above background by a small increment, were found at 16 positions throughout the region of ribosome contact. These substitutions lengthened the Shine-Dalgarno complementarity or changed the G and C residues present in the wild-type site to A or T. Significant activity was not observed unless a strong Shine-Dalgarno sequence and a number of the up-mutations were present together. The nature and distribution of the substitutions and their agreement with the known preferences for nucleotides in initiation sites provide evidence that the VII site's major defect is its primary sequence overall. It appears to lack the specialized sequence required to bind free 30 S ribosomes, and thus depends on the translational coupling process to give it limited activity.     

Molecular cloning and nucleotide sequence of the HU-1 gene of Escherichia coli

Summary The Escherichia coli HU-1 was cloned by use of mixed synthetic oligonucleotides (17-mer) predicted from a portion of its amino acid sequence. The amino acid sequence of the HU-1 protein deduced from the nucleotide sequence is in good agreement with the published sequence. The nucleotide sequence has a possible promoter and a typical ribosomal binding site upstream from the translational initiation codon (GUG) of the HU-1 gene.     

Cloning and sequencing of the HU-2 gene of Escherichia coli

Summary The Escherichia coli HU-2 gene was cloned using a DNA fragment from the HU-1 gene as a probe. The amino acid sequence of the HU-2 protein deduced from the nucleotide sequence is in good agreement with the published sequence. The nucleotide sequence has a possible promoter and a typical ribosomal binding site upstream of the translation initiation codon (AUG) and a possible rhoindependent terminater site downstream of the termination codon (UAA) of the gene.     

The influence of ribosome-binding-site elements on translational efficiency in Bacillus subtilis and Escherichia coli in vivo

A method is described to determine simultaneously the effect of any changes in the ribosome-binding site (RBS) of mRNA on translational efficiency in Bacillus subtilis and Escherichia coli in vivo. The approach was used to analyse systematically the influence of spacing between the Shine-Dalgarno sequence and the initiation codon, the three different initiation codons, and RBS secondary structure on translational yields in the two organisms. Both B. subtilis and E. coli exhibited similar spacing optima of 7-9 nucleotides. However, B. subtilis translated messages with spacings shorter than optimal much less efficiently than E. coli. In both organisms, AUG was the preferred initiation codon by two- to threefold. In E. coli GUG was slightly better than UUG while in B. subtilis UUG was better than GUG. The degree of emphasis placed on initiation codon type, as measured by translational yield, was dependent on the strength of the Shine-Dalgarno interaction in both organisms. B. subtilis was also much less able to tolerate secondary structure in the RBS than E. coli. While significant differences were found between the two organisms in the effect of specific RBS elements on translation, other mRNA components in addition to those elements tested appear to be responsible, in part, for translational species specificity. The approach described provides a rapid and systematic means of elucidating such additional determinants.     

Translation initiation in Escherichia coli: sequences within the ribosome-binding site

The translational roles of the Shine-Dalgarno sequence, the initiation codon, the space between them, and the second codon have been studied. The Shine-Dalgarno sequence UAAGGAGG initiated translation roughly four times more efficiently than did the shorter AAGGA sequence. Each Shine-Dalgarno sequence required a minimum distance to the initiation codon in order to drive translation; spacing, however, could be rather long. Initiation at AUG was more efficient than at GUG or UUG at each spacing examined; initiation at GUG was only slightly better than UUG. Translation was also affected by residues 3' to the initiation codon. The second codon can influence the rate of initiation, with the magnitude depending on the initiation codon. The data are consistent with a simple kinetic model in which a variety of rate constants contribute to the process of translation initiation.     

RNA primary sequence or secondary structure in the translational initiation region controls expression of two variant interferon-beta genes in Escherichia coli

Efficient expression in Escherichia coli (E. coli) of the human interferon-beta gene (IFN-beta) gene and of a chemically synthesized IFN-beta gene variant (506 base pairs; synIFN-beta) adapted to the E. coli codon usage, both fused to the E. coli atpE ribosome-binding site, is controlled either by primary sequence or by mRNA secondary-structure in the translational initiation region. High level expression of the natural human atpE/IFN-beta gene fusion is governed by the nucleotide composition preceding the initiator codon AUG. A single U----C exchange in the -2 or -1 position preceding the initiator codon AUG reduces the translational efficiency from 18% of total cellular protein to only 8% or 4%, respectively, while both U----C substitutions reduce IFN-beta expression below 1%. These sequence alterations interfere with efficient ribosome binding as revealed by toeprinting. They provide further evidence for the influence of the anticodon-flanking regions of tRNA(fMet) upon the initiation rate of translation. In contrast, translation of the synthetic variant atpE/synIFN-beta gene fusion is controlled by a moderately stable stem-loop structure (delta G = -4 kcal/mol; 37 degrees C) located within the coding region and overlapping the 30 S ribosomal subunit attachment site. That the stability of the hairpin interferes with the initiation of translation is inferred from site-directed mutagenesis and toeprint analyses. mRNA half-life in these variants is positively correlated with the rate of translation and involves two major endonucleolytic cleavage site 5'-upstream of the Shine-Dalgarno region.     

Local absence of secondary structure permits translation of mRNAs that lack ribosome-binding sites

The initiation of translation is a fundamental and highly regulated process in gene expression. Translation initiation in prokaryotic systems usually requires interaction between the ribosome and an mRNA sequence upstream of the initiation codon, the so-called ribosome-binding site (Shine-Dalgarno sequence). However, a large number of genes do not possess Shine-Dalgarno sequences, and it is unknown how start codon recognition occurs in these mRNAs. We have performed genome-wide searches in various groups of prokaryotes in order to identify sequence elements and/or RNA secondary structural motifs that could mediate translation initiation in mRNAs lacking Shine-Dalgarno sequences. We find that mRNAs without a Shine-Dalgarno sequence are generally less structured in their translation initiation region and show a minimum of mRNA folding at the start codon. Using reporter gene constructs in bacteria, we also provide experimental support for local RNA unfoldedness determining start codon recognition in Shine-Dalgarno--independent translation. Consistent with this, we show that AUG start codons reside in single-stranded regions, whereas internal AUG codons are usually in structured regions of the mRNA. Taken together, our bioinformatics analyses and experimental data suggest that local absence of RNA secondary structure is necessary and sufficient to initiate Shine-Dalgarno--independent translation. Thus, our results provide a plausible mechanism for how the correct translation initiation site is recognized in the absence of a ribosome-binding site.     

Translation of the leaderless Caulobacter dnaX mRNA.

The expression of the Caulobacter crescentus homolog of dnaX, which in Escherichia coli encodes both the gamma and tau subunits of the DNA polymerase III holoenzyme, is subject to cell cycle control. We present evidence that the first amino acid in the predicted DnaX protein corresponds to the first codon in the mRNA transcribed from the dnaX promoter; thus, the ribosome must recognize the mRNA at a site downstream of the start codon in an unusual but not unprecedented fashion. Inserting four bases in front of the AUG at the 5' end of dnaX mRNA abolishes translation in the correct frame. The sequence upstream of the translational start site shows little homology to the canonical Shine-Dalgarno ribosome recognition sequence, but the region downstream of the start codon is complementary to a region of 16S rRNA implicated in downstream box recognition. The region downstream of the dnaX AUG, which is important for efficient translation, exhibits homology with the corresponding region from the Caulobacter hemE gene adjacent to the replication origin. The hemE gene also appears to be translated from a leaderless mRNA. Additionally, as was found for hemE, an upstream untranslated mRNA also extends into the dnaX coding sequence. We propose that translation of leaderless mRNAs may provide a mechanism by which the ribosome can distinguish between productive and nonproductive templates.     

Biosynthesis of osteogenic growth peptide via alternative translational initiation at AUG85 of histone H4 mRNA.

The osteogenic growth peptide (OGP) is an extracellular mitogen identical to the histone H4 (H4) COOH-terminal residues 90-103, which regulates osteogenesis and hematopoiesis. By Northern analysis, OGP mRNA is indistinguishable from H4 mRNA. Indeed, cells transfected with a construct encoding [His102]H4 secreted the corresponding [His13]OGP. These results suggest production of OGP from H4 genes. Cells transfected with H4-chloramphenicol acetyltransferase (CAT) fusion genes expressed both "long" and "short" CAT proteins. The short CAT was retained following an ATG --> TTG mutation of the H4 ATG initiation codon, but not following mutation of the in-frame internal ATG85 codon, which, unlike ATG1, resides within a perfect context for translational initiation. These results suggest that a PreOGP is translated starting at AUG85. The translational initiation at AUG85 could be inhibited by optimizing the nucleotide sequence surrounding ATG1 to maximally support upstream translational initiation, thus implicating leaky ribosomal scanning in usage of the internal AUG. Conversion of the predicted PreOGP to OGP was shown in a cell lysate system using synthetic [His102]H4-(85-103) as substrate. Together, our results demonstrate that H4 gene expression diverges at the translational level into the simultaneous parallel production of both H4, a nuclear structural protein, and OGP, an extracellular regulatory peptide.     

Multiple elements required for translation of plastid atpB mRNA lacking the Shine-Dalgarno sequence

The mechanism of translational initiation differs between prokaryotes and eukaryotes. Prokaryotic mRNAs generally contain within their 5′-untranslated region (5′-UTR) a Shine-Dalgarno (SD) sequence that serves as a ribosome-binding site. Chloroplasts possess prokaryotic-like translation machinery, and many chloroplast mRNAs have an SD-like sequence, but its position is variable. Tobacco chloroplast atpB mRNAs contain no SD-like sequence and are U-rich in the 5′-UTR (−20 to −1 with respect to the start codon). In vitro translation assays with mutated mRNAs revealed that an unstructured sequence encompassing the start codon, the AUG codon and its context are required for translation. UV crosslinking experiments showed that a 50 kDa protein (p50) binds to the 5′-UTR. Insertion of an additional initiation region (SD-sequence and AUG) in the 5′-UTR, but not downstream, arrested translation from the authentic site; however, no inhibition was observed by inserting only an AUG triplet. We hypothesize for translational initiation of the atpB mRNA that the ribosome enters an upstream region, slides to the start codon and forms an initiation complex with p50 and other components.