Escherichia coli RNase D: sequencing of the rnd structural gene and purification of the overexpressed protein.

We have determined the nucleotide sequence of a 1.4-kb-pair fragment of the E. coli chromosome that carries the complete rnd gene encoding RNase D, a putative tRNA processing enzyme. The coding region of rnd extends for a total of 1128 nucleotides beginning at an initiator UUG codon and terminating at a UAA codon, and encodes a 375-amino acid polypeptide of 42,679 daltons, consistent with the known size of RNase D. A rapid purification procedure was developed for isolation of RNase D from strains overexpressing the enzyme. The N-terminal sequence and the amino acid composition of the homogenous protein were in excellent agreement with those derived from the sequence of the rnd gene.     

The chloroplast infA gene with a functional UUG initiation codon

All chloroplast genes reported so far possess ATG start codons and sometimes GTGs as an exception. Sequence alignments suggested that the chloroplast infA gene encoding initiation factor 1 in the green alga Chlorella vulgaris has TTG as a putative initiation codon. This gene was shown to be transcribed by RT-PCR analysis. The infA mRNA was translated accurately from the UUG codon in a tobacco chloroplast in vitro translation system. Mutation of the UUG codon to AUG increased translation efficiency approximately 300-fold. These results indicate that the UUG is functional for accurate translation initiation of Chlorella infA mRNA but it is an inefficient initiation codon.     

Translational initiation at the coat-protein gene of phage MS2: native upstream RNA relieves inhibition by local secondary structure

Maximal translation of the coat-protein gene from RNA bacteriophage MS2 requires a contiguous stretch of native MS2 RNA that extends hundreds of nucleotides upstream from the translational start site. Deletion of these upstream sequences from MS2 cDNA plasmids results in a 30-fold reduction of translational efficiency. By site-directed mutagenesis, we show that this low level of expression is caused by a hairpin structure centred around the initiation codon. When this hairpin is destabilized by the introduction of mismatches, expression from the truncated messenger increases 20-fold to almost the level of the full-length construct. Thus, the translational effect of hundreds of upstream nucleotides can be mimicked by a single substitution that destabilizes the structure. The same hairpin is also present in full-length MS2 RNA, but there it does not Impair ribosome binding. Apparently, the upstream RNA somehow reduces the inhibitory effect of the structure on translational initiation. The upstream MS2 sequence does not stimulate translation when cloned in front of another gene, nor can unrelated RNA segments activate the coat-protein gene. Several possible mechanisms for the activation are discussed and a function in gene regulation of the phage is suggested.     

Translation of phage f1 gene VII occurs from an inherently defective initiation site made functional by coupling

Expression of the filamentous phage f1 gene VII is shown to be translationally coupled to that of the upstream gene V. Fusions of the gene VII initiation site to the lacZ coding region were used to determine that initiation at the VII site is completely dependent on the process of translation having proceeded up to a stop codon immediately upstream from the VII site. Coupled expression from the VII site was found to be inefficient, proportional to the level of upstream translation, and very sensitive to the distance from the functional upstream stop codon. Independent expression from the VII site was not observed, even in a deletion series designed to remove potentially masking RNA structure. On the basis of the VII site's dissimilarity to ribosome binding site sequences and its properties overall, we suggest that it inherently lacks the features required for independent recognition by ribosomes, and acquires the ability to initiate synthesis of gene VII protein by virtue of the coupling process.     

Influences on gene expression in vivo by a Shine-Dalgarno sequence

The Shine-Dalgarno (SD+: 5'-AAGGAGG-3') sequence anchors the mRNA by base pairing to the 16S rRNA in the small ribosomal subunit during translation initiation. We have here compared how an SD+ sequence influences gene expression, if located upstream or downstream of an initiation codon. The positive effect of an upstream SD+ is confirmed. A downstream SD+ gives decreased gene expression. This effect is also valid for appropriately modified natural Escherichia coli genes. If an SD+ is placed between two potential initiation codons, initiation takes place predominantly at the second start site. The first start site is activated if the distance between this site and the downstream SD+ is enlarged and/or if the second start site is weakened. Upstream initiation is eliminated if a stable stem-loop structure is placed between this SD+ and the upstream start site. The results suggest that the two start sites compete for ribosomes that bind to an SD+ located between them. A minor positive contribution to upstream initiation resulting from 3' to 5' ribosomal diffusion along the mRNA is suggested. Analysis of the E. coli K12 genome suggests that the SD+ or SD-like sequences are systematically avoided in the early coding region suggesting an evolutionary significance.     

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.     

An unstructured mRNA region and a 5' hairpin represent important elements of the E. coli translation initiation signal determined by using the bacteriophage T7 gene 1 translation start site.

Gene 1 of bacteriophage T7 early region--the RNA polymerase gene--is very actively translated during the infectious cycle of this phage. A 29 base pair fragment of its ribosome binding site containing the initiation triplet, the Shine-Dalgarno sequence (S-D), 10 nucleotides (nt) upstream and 6 nt downstream of these central elements was cloned into a vector to control the expression of the mouse dihydrofolate reductase gene (dhfr). Although all essential parts of this translation initiation region (TIR) should be present, this fragment showed only very low activity. Computer analysis revealed a potentially inhibitory hairpin binding the S-D sequence into its stem base paired to vector-derived upstream sequences. Mutational alterations demonstrated that this hairpin was not responsible for the low activity. However, addition of 21 nt of the T7 gene 1 upstream sequence to the 29 base pair fragment were capable of increasing the translational efficiency by one order of magnitude. Computer analysis of this sequence, including nucleotide shuffling, revealed that it contains a highly unstructured region lacking mRNA secondary structures but with a hairpin at its 5' end, here formed solely by T7 sequences. There was not much difference in activity whether the mRNA included or lacked vector-derived sequences upstream of the hairpin. Such highly unstructured mRNA regions were found in all very efficiently expressed T7 genes without any obvious sequence homologies. The delta G values of these regions were higher, i.e. potential secondary structural elements were fewer, than in TIR of genes from E. coli. This is likely due to the fact that T7 as a lytic phage is relying for successful infection on much stronger signals which a cell cannot afford because of the indispensable balanced equilibria of its interdependent biochemical processes. When the 5' ends of efficient T7 gene mRNA are formed by the action of RNase III they generally start with an unstructured region. Efficiently expressed T7 genes within a polycistronic mRNA, however, always contain a hairpin preceding the structure free sequence. We suggest that the formation of this 5' hairpin is releasing enough energy to keep the unstructured regions free of secondary RNA structures for sufficient time to give ribosomes and factors a good chance for binding to the TIR. In addition, sequences further downstream of the start codon give rise to an additional increase in efficiency of the TIR by almost two orders of magnitude.     

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.