Antagonistic signals within the COX2 mRNA coding sequence control its translation in Saccharomyces cerevisiae mitochondria

Translation of the mitochondrially coded COX2 mRNA within the organelle in yeast produces the precursor of Cox2p (pre-Cox2p), which is processed and assembled into cytochrome c oxidase. The mRNA sequence of the first 14 COX2 codons, specifying the pre-Cox2p leader peptide, was previously shown to contain a positively acting element required for translation of a mitochondrial reporter gene, ARG8(m), fused to the 91st codon of COX2. Here we show that three relatively short sequences within the COX2 mRNA coding sequence, or structures they form in vivo, inhibit translation of the reporter in the absence of the positive element. One negative element was localized within codons 15 to 25 and shown to function at the level of the mRNA sequence, whereas two others are within predicted stem-loop structures formed by codons 22-44 and by codons 46-74. All three of these inhibitory elements are antagonized in a sequence-specific manner by reintroduction of the upstream positive-acting sequence. These interactions appear to be independent of 5'- and 3'-untranslated leader sequences, as they are also observed when the same reporter constructs are expressed from the COX3 locus. Overexpression of MRS2, which encodes a mitochondrial magnesium carrier, partially suppresses translational inhibition by each isolated negatively acting element, but does not suppress them in combination. We hypothesize that interplay among these signals during translation in vivo may ensure proper timing of pre-Cox2p synthesis and assembly into cytochrome c oxidase.     

Mutations affecting pseudoknot control of the replication of B group plasmids.

The translational initiation region of the mRNA for the replication initiation protein (RepA) of pMU720 is predicted to be sequestered in an inhibitory secondary structure designated stem-loop III. Activation of repA translation requires both the disruption of stem-loop III by ribosomes involved in the translation and termination of the leader peptide RepB and the formation of a pseudoknot, a tertiary RNA structure. Disruption of stem-loop III by site-directed mutagenesis was found to be insufficient to allow high repA expression in the absence of pseudoknot formation, indicating that the pseudoknot acts as an enhancer of repA translation. Furthermore, extending the length of the leader peptide RepB and changing the distance between the pseudoknot and repA Shine-Dalgarno sequence were found to have major effects on the translation of repA.     

The translational regulation of threonyl-tRNA synthetase. Functional relationship between the enzyme, the cognate tRNA and the ribosome

The E. coli threonyl-tRNA synthetase gene is negatively autoregulated at the translational level by a direct binding of the enzyme to the leader region of the thrS mRNA. This region folds in four well-defined domains. The enzyme binds to the leader at two major sites: the first is a stem-loop structure located in domain II upstream of the translational initiation site (domain I) which shares structural analogies with the anticodon arm of several tRNA(Thr) isoacceptors. The second site corresponds to a stable stem-loop structure located in domain IV. Both sites are separated by a large unpaired region (domain III). In vivo and in vitro experiments show that the structural integrity of both sites is required for the regulatory process. The binding of the enzyme to its mRNA target site represses its translation by preventing the ribosome from binding to its attachment site. tRNA(Thr) suppresses this inhibitory effect by displacing the mRNA from the enzyme at both the upstream stem-loop structure and the tRNA-like anticodon arm.     

SLIP1, a factor required for activation of histone mRNA translation by the stem-loop binding protein

Replication-dependent histone mRNAs are the only eukaryotic cellular mRNAs that are not polyadenylated, ending instead in a conserved stem-loop. The 3′ end of histone mRNA is required for histone mRNA translation, as is the stem-loop binding protein (SLBP), which binds the 3′ end of histone mRNA. We have identified five conserved residues in a 15-amino-acid region in the amino-terminal portion of SLBP, each of which is required for translation. Using a yeast two-hybrid screen, we identified a novel protein, SLBP-interacting protein 1 (SLIP1), that specifically interacts with this region. Mutations in any of the residues required for translation reduces SLIP1 binding to SLBP. The expression of SLIP1 in Xenopus oocytes together with human SLBP stimulates translation of a reporter mRNA ending in the stem-loop but not a reporter with a poly(A) tail. The expression of SLIP1 in HeLa cells also stimulates the expression of a green fluorescent protein reporter mRNA ending in a stem-loop. RNA interference-mediated downregulation of endogenous SLIP1 reduces the rate of translation of endogenous histone mRNA and also reduces cell viability. SLIP1 may function by bridging the 3′ end of the histone mRNA with the 5′ end of the mRNA, similar to the mechanism of translation of polyadenylated mRNAs.     

Role for cis-Acting RNA Sequences in the Temperature-Dependent Expression of the Multiadhesive Lig Proteins in Leptospira interrogans

The spirochete Leptospira interrogans causes a systemic infection that provokes a febrile illness. The putative lipoproteins LigA and LigB promote adhesion of Leptospira to host proteins, interfere with coagulation, and capture complement regulators. In this study, we demonstrate that the expression level of the LigA and LigB proteins was substantially higher when L. interrogans proliferated at 37°C instead of the standard culture temperature of 30°C. The RNA comprising the 175-nucleotide 5′ untranslated region (UTR) and first six lig codons, whose sequence is identical in ligA and ligB, is predicted to fold into two distinct stem-loop structures separated by a single-stranded region. The ribosome-binding site is partially sequestered in double-stranded RNA within the second structure. Toeprint analysis revealed that in vitro formation of a 30S-tRNA^fMet-mRNA ternary complex was inhibited unless a 5′ deletion mutation disrupted the second stem-loop structure. To determine whether the lig sequence could mediate temperature-regulated gene expression in vivo, the 5′ UTR and the first six codons were inserted between the Escherichia coli l-arabinose promoter and bgaB (β-galactosidase from Bacillus stearothermophilus) to create a translational fusion. The lig fragment successfully conferred thermoregulation upon the β-galactosidase reporter in E. coli. The second stem-loop structure was sufficient to confer thermoregulation on the reporter, while sequences further upstream in the 5′ UTR slightly diminished expression at each temperature tested. Finally, the expression level of β-galactosidase was significantly higher when point mutations predicted to disrupt base pairs in the second structure were introduced into the stem. Compensatory mutations that maintained base pairing of the stem without restoring the wild-type sequence reinstated the inhibitory effect of the 5′ UTR on expression. These results indicate that ligA and ligB expression is limited by double-stranded RNA that occludes the ribosome-binding site. At elevated temperatures, the ribosome-binding site is exposed to promote translation initiation.     

Regulation of zinc-responsive Slc39a5 (Zip5) translation is mediated by conserved elements in the 3′-untranslated region

Translation of the basolateral zinc transporter ZIP5 is repressed during zinc deficiency but Zip5 mRNA remains associated with polysomes and can be rapidly translated when zinc is repleted. Herein, we examined the mechanisms regulating translation of Zip5. The 3′-untranslated region (UTR) of Zip5 mRNA is well conserved among mammals and is predicted by mFOLD to form a very stable stem-loop structure. Three algorithms predict this structure to be flanked by repeated seed sites for miR-328 and miR-193a. RNAse footprinting supports the notion that a stable stem-loop structure exists in this 3′-UTR and electrophoretic mobility shift assays detect polysomal protein(s) binding specifically to the stem-loop structure in the Zip5 3′-UTR. miR-328 and miR-193a are expressed in tissues known to regulate Zip5 mRNA translation in response to zinc availability and both are polysome-associated consistent with Zip5 mRNA localization. Transient transfection assays using native and mutant Zip5 3′-UTRs cloned 3′ to luciferase cDNA revealed that the miRNA seed sites and the stem-loop function together to augment translation of Zip5 mRNA when zinc is replete.     

Identification and Characterization of the Functional Elements within the Tobacco Etch Virus 5′ Leader Required for Cap-Independent Translation

Translation in plants is highly cap dependent, and the only plant mRNAs known to naturally lack a cap structure (m(7)GpppN) are viral in origin. The genomic RNA of tobacco etch virus (TEV), a potyvirus that belongs to the picornavirus superfamily, is a polyadenylated mRNA that is naturally uncapped and yet is a highly competitive mRNA during translation. The 143-nucleotide 5' leader is responsible for conferring cap-independent translation even on reporter mRNAs. We have carried out a deletion analysis of the TEV 5' leader to identify the elements responsible for its regulatory function and have identified two centrally located cap-independent regulatory elements (CIREs) that promote cap-independent translation. The introduction of a stable stem-loop structure upstream of each element demonstrated that CIRE-1 is less 5' end dependent in function than CIRE-2. In a dicistronic mRNA, the presence of the TEV 5' leader sequence in the intercistronic region increased expression of the second cistron, suggesting that the viral sequence can function in a 5'-distal position. Interestingly, the introduction of a stable stem-loop upstream of the TEV leader sequence or upstream of either CIRE in dicistronic constructs markedly increased their regulatory function. These data suggest that the TEV 5' leader contains two elements that together promote internal initiation but that the function of one element, in particular, is facilitated by proximity to the 5' end.     

mRNA sequences influencing translation and the selection of AUG initiator codons in the yeast Saccharomyces cerevisiae

The secondary structure and sequences influencing the expression and selection of the AUG initiator codon in the yeast Saccharomyces cerevisiae were investigated with two fused genes, which were composed of either the CYC7 or CYC1 leader regions, respectively, linked to the lacZ coding region. In addition, the strains contained the upf1-Δ disruption, which stabilized mRNAs that had premature termination codons, resulting in wild-type levels. The following major conclusions were reached by measuring β-galactosidase activities in yeast strains having integrated single copies of the fused genes with various alterations in the 89 and 38 nucleotide-long untranslated CYC7 and CYC1 leader regions, respectively. The leader region adjacent to the AUG initiator codon was dispensable, but the nucleotide preceding the AUG initiator at position ?3 modified the efficiency of translation by less than twofold, exhibiting an order of preference A>G>C>U. Upstream out-of-frame AUG triplets diminished initiation at the normal site, from essentially complete inhibition to approximately 50% inhibition, depending on the position of the upstream AUG triplet and on the context (?3 position nucleotides) of the two AUG triplets. In this regard, complete inhibition occurred when the upstream and downstream AUG triplets were closer together, and when the upstream and downstream AUG triplets had, respectively, optimal and suboptimal contexts. Thus, leaky scanning occurs in yeast, similar to its occurrence in higher eukaryotes. In contrast, termination codons between two AUG triplets causes reinitiation at the downstream AUG in higher eukaryotes, but not generally in yeast. Our results and the results of others with GCN4 mRNA and its derivatives indicate that reinitiation is not a general phenomenon in yeast, and that special sequences are required.     

Plant ribosome shunting in vitro.

It has been proposed that cauliflower mosaic virus 35S RNA with its 600 nt long leader uses an unusual translation process (the translational shunt). A wheat germ in vitro translation assay was used to improve the study of this mechanism. Deletions, the introduction of stable stem-loop structures, and the inhibitory effect of antisense oligonucleotides on gene expression were used to determine the roles of various parts of the leader. It was found that the 5'- and 3'-ends of the leader are absolutely required for translation whereas the middle part is apparently dispensable. These results confirm the data already reported from transient expression experiments with protoplasts. However, the in vitro data suggest in contrast to protoplast experiments that only two relatively short regions at both ends, approximately 100 nt each, are required. The in vitro system provides tools for further studying the shunt model at the molecular level and for examining the involvement of proteins in this mechanism. Shunting was also found to occur with the rice tungro bacilliform virus leader. As wheat is neither a host plant of cauliflower mosaic virus nor rice tungro bacilliform virus, the shunt seems to be host independent, a finding that deviates from earlier studies in protoplasts.     

Binding of LARP6 to the Conserved 5′ Stem-Loop Regulates Translation of mRNAs Encoding Type I Collagen

Type I collagen is the most abundant protein in the human body, produced by folding of two α1(I) polypeptides and one α2(I) polypeptide into the triple helix. A conserved stem-loop structure is found in the 5′ untranslated region of collagen mRNAs, encompassing the translation start codon. We cloned La ribonucleoprotein domain family member 6 (LARP6) as the protein that binds the collagen 5′ stem-loop in a sequence-specific manner. LARP6 has a distinctive bipartite RNA binding domain not found in other members of the La superfamily. LARP6 interacts with the two single-stranded regions of the 5′ stem-loop. The K_d for binding of LARP6 to the 5′ stem-loop is 1.4 nM. LARP6 binds the 5′ stem-loop in both the nucleus and the cytoplasm. In the cytoplasm, LARP6 does not associate with polysomes; however, overexpression of LARP6 blocks ribosomal loading on collagen mRNAs. Knocking down LARP6 by small interfering RNA also decreased polysomal loading of collagen mRNAs, suggesting that it regulates translation. Collagen protein is synthesized at discrete regions of the endoplasmic reticulum. Using collagen-GFP (green fluorescent protein) reporter protein, we could reproduce this focal pattern of synthesis, but only when the reporter was encoded by mRNA with the 5′ stem-loop and in the presence of LARP6. When the reporter was encoded by mRNA without the 5′ stem-loop, or in the absence of LARP6, it accumulated diffusely throughout the endoplasmic reticulum. This indicates that LARP6 activity is needed for focal synthesis of collagen polypeptides. We postulate that the LARP6-dependent mechanism increases local concentration of collagen polypeptides for more efficient folding of the collagen heterotrimer.     

Translation attenuation regulation of chloramphenicol resistance in bacteria — a review

The chloramphenicol (Cm)-inducible cat and cmlA genes are regulated by translation attenuation, a regulatory device that modulates mRNA translation. In this form of gene regulation, translation of the Cm^R coding sequence is prevented by mRNA secondary structure that sequesters its ribosome-binding site (RBS). A translated leader of nine codons precedes the secondary structure, and induction results when a ribosome becomes stalled at a specific site in the leader. Here we demonstrate that the site of ribosome stalling in the leader is selected by a cis effect of the nascent leader peptide on its translating ribosome.     

Shine-Dalgarno-like sequences are not required for translation of chloroplast mRNAs in Chlamydomonas reinhardtii chloroplasts or in Escherichia coli

Initiation of translation in Escherichia coli and related eubacteria involves well-defined interactions between a conserved Shine-Dalgarno (SD) sequence immediately upstream of the initiation codon in the mRNA leader and an equally conserved anti-SD sequence at the 3′ end of the 16S rRNA. SD-like sequences found in the leaders of many, but not all, mRNAs from cyanobacteria and chloroplasts are hypervariable in location, size, and base composition compared to those in E. coli, while anti-SD sequences in the respective 16S rRNAs remain highly conserved. We have examined the function of the SD-like sequences found in the leaders of four chloroplast genes of the green alga Chlamydomonas reinhardtii using replacement mutagenesis to eliminate complementarity with the anti-SD sequences and insertion of canonical SD sequences (GGAGG) at positions −9 to −5 relative to the initiation codon. Promoter-leader regions of the atpB, atpE, rps4, and rps7 genes representing the diversity of chloroplast SD-like sequences were fused to aadA and uidA reporter genes encoding spectinomycin resistance and GUS activity respectively. Analysis of chloroplast transformants of C. reinhardtii and transformants of E. coli carrying the wild-type and mutant reporter constructs revealed that mutagenic replacement of the putative SD sequences had no effect on the expression of either the aadA or uidA reporter genes. Chloroplast transformants with the canonical SD sequence also showed no differences in reporter gene expression, whereas expression of the reporter genes was increased by 10 to 30% in the E. coli transformants. Collectively our results suggest that even though SD-dependent initiation predominates in E. coli, this bacterium also has the capacity to initiate translation by an SD-independent mechanism. In contrast, plant chloroplasts, and very probably their cyanobacterial ancestors, appear to have adopted the SD-independent mechanism for translational initiation of most mRNAs. Received: 8 July 1997 / Accepted: 9 September 1997     

Close vicinity of IS1 integration sites in the leader sequence of the gal operon of E. coli.

Summary Four insertions of IS1 in the leader sequence of the gal operon of E. coli have been analysed.Two of them occur at the same position, but in opposite orientations. The other two are inserted one nucleotide to one side and four nucleotides to the other side, respectively.In each case, nine base pairs of the leader sequence of the gal operon are duplicated directly, and are found flanking the termini of IS1 at its junction with the gal operon. These repeated sequences differ from each other as expected from the different insertion sites.