A possible contribution of mRNA secondary structure to translation initiation efficiency in Lactococcus lactis

Gene expression signals derived from Lactococcus lactis were linked to lacZ-fused genes with different 5'-nucleotide sequences. Computer predictions of mRNA secondary structure were combined with lacZ expression studies to direct base-substitutions that could possibly influence gene expression. Mutations were made such that the DNA sequence upstream of the ATG start codon was not changed. Moreover, care was taken that the substitutions, which were all within the first six codons, neither affected the amino acid sequence of the gene product nor introduced codons rarely used in L. lactis. The results suggest that mRNA secondary structure contributes to the efficiency of translation initiation in L. lactis.     

Correlation between the effectiveness of translation initiation and secondary structure of mRNA in the hybrid gene cro-lacIZ

A set of plasmids containing short DNA deletions in the N-part of cro-lacIZ coding zone as constructed using Bal31 nuclease. Development of models of mRNA secondary structure was carried out stepwise beginning from the 5'-end by taking into consideration the hairpins first formed during mRNA synthesis. Comparison of the results of mRNA secondary structure determination and protein production analysis demonstrated a correlation between the efficiency of translation initiation and the appearance of a single-stranded region upon disruption of the mRNA local secondary structure in the translation initiation zone generated by ribosomes. These results confirm the suggestion of the central role played by the Shine-Dalgarno sequence in the generation of a single-stranded region. Some plasmids from the set are supposed to determine protein synthesis by a translation reinitiation mechanism in the absence of Shine--Dalgarno interaction. In this case, correlation between reinitiation efficiency and local disruption of the mRNA secondary structure by the terminating ribosome was also observed. The terminating ribosome that forms the single-stranded region near the initiation codon fulfils the major function of the Shine--Dalgarno interaction. In addition, the possible effect of another mRNA secondary structure region on the translation initiation efficiency is discussed.     

Effect of mRNA secondary structure on the efficiency of translational initiation by eukaryotic ribosomes

We have used site-directed mutagenesis to determine whether the structural context surrounding the AUG triplet influences its ability to be selected as an initiation codon by the eukaryotic preinitiation complex. AUG triplets were introduced in a loop and stem structure naturally occurring at the midpoint of the 129-nucleotides-long 5'-untranslated region of the porcine proopiomelanocortin mRNA; one AUG triplet was inserted in the loop while another was inserted in the stem of the hairpin structure. The proopiomelanocortin cDNA and the mutant cDNAs were inserted downstream from the early promoter of an expression vector derived from simian virus 40 (SV40) and transfected into monkey kidney COS-1 cells. Analysis of the proopiomelanocortin-related peptides present in the culture medium 72 h after transfection revealed that both mutant cDNAs direct the synthesis of more proopiomelanocortin than the non-mutant cDNA. The increased translational efficiency observed with both mutants is probably due to the decreased secondary structures of the shortened 5'-untranslated region. In addition, comparison of the two mutants indicates that the mutant mRNA with the AUG triplet inserted in the loop region of the hairpin structure directs the synthesis of approximately 75% more proopiomelanocortin than the mutant mRNA with the AUG triplet inserted in the stem region of the same hairpin structure, supporting a role for the structural context in the efficiency of translational initiation.     

mRNA secondary structure modulates translation of Tat-dependent formate dehydrogenase N

Formate dehydrogenase N (FDH-N) of Escherichia coli is a membrane-bound enzyme comprising FdnG, FdnH, and FdnI subunits organized in an (alphabetagamma)3 configuration. The FdnG subunit carries a Tat-dependent signal peptide, which localizes the protein complex to the periplasmic side of the membrane. We noted that substitution of the first arginine (R5) in the twin arginine signal sequence of FdnG for a variety of other amino acids resulted in a dramatic (up to 60-fold) increase in the levels of protein synthesized. Bioinformatic analysis suggested that the mRNA specifying the first 17 codons of fdnG forms a stable stem-loop structure. A detailed mutational analysis has demonstrated the importance of this mRNA stem-loop in modulating FDH-N translation.     

Rapid 40S scanning and its regulation by mRNA structure during eukaryotic translation initiation

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Sulfolobus solfataricus translation initiation factor 1 stimulates translation initiation complex formation

The eukaryotic translation initiation factor 1 binds to the ribosome during translation initiation. It is instrumental for initiator-tRNA and mRNA binding, and has a function in selection of the authentic start codon. Here, we show that the archaeal homolog aIF1 has analogous functions. The aIF1 protein of the archaeon Sulfolobus solfataricus is bound to the small ribosomal subunit during translation initiation and accelerates binding of initiator-tRNA and mRNA to the ribosome. Accordingly, aIF1 stimulated translation of an mRNA in a S. solfataricus in vitro translation system. Moreover, this study suggested that the C terminus of the factor is of relevance for its function.     

The cryo-EM structure of a translation initiation complex from Escherichia coli

The 70S ribosome and its complement of factors required for initiation of translation in E. coli were purified separately and reassembled in vitro with GDPNP, producing a stable initiation complex (IC) stalled after 70S assembly. We have obtained a cryo-EM reconstruction of the IC showing IF2*GDPNP at the intersubunit cleft of the 70S ribosome. IF2*GDPNP contacts the 30S and 50S subunits as well as fMet-tRNA(fMet). IF2 here adopts a conformation radically different from that seen in the recent crystal structure of IF2. The C-terminal domain of IF2 binds to the single-stranded portion of fMet-tRNA(fMet), thereby forcing the tRNA into a novel orientation at the P site. The GTP binding domain of IF2 binds to the GTPase-associated center of the 50S subunit in a manner similar to EF-G and EF-Tu. Additionally, we present evidence for the localization of IF1, IF3, one C-terminal domain of L7/L12, and the N-terminal domain of IF2 in the initiation complex.     

Ribosomal proteins cross-linked to the initiator AUG codon of a mRNA in the translation initiation complex by UV-irradiation

Eukaryotic ribosomal proteins constituting the binding site for the initiator codon AUG on the ribosome at the translation initiation step were investigated by UV-induced cross-linking between protein and mRNA. The 80S-initiation complex was formed in a rabbit reticulocyte cell-free system in the presence of sparsomycin with radiolabeled Omega-fragment as a template, which was a 73-base 5'-leader sequence of tobacco mosaic virus RNA having AUG at the extreme 3'-terminal end and extended with 32pCp. Two radioactive peaks were sedimented by sucrose gradient centrifugation, one being the 80S initiation complex formed at the 3'-terminal AUG codon, and the other presumably a "disome" with an additional 80S ribosome bound at an upstream AUU codon, formed when Omega-fragment was incubated with sparsomycin [Filipowicz and Henni (1979) Proc. Natl. Acad. Sci. USA 76, 3111-3115]. Cross-links between ribosomal proteins and the radiolabeled Omega-fragment were induced in situ by UV-irradiation at 254 nm. After extensive nuclease digestion of the complexes, ribosomal proteins were separated by two-dimensional gel electrophoresis. Autoradiography identified the proteins S7, S10, S25, S29, and L5 of the 80S initiation complex and S7, S25, S29 and L5 of that in the disome as 32P-labeled proteins. Together with the results of cross-linking experiments of other investigators and recently solved crystal structures of prokaryotic ribosomes, the spatial arrangement of eukaryotic ribosomal proteins at the AUG-binding domain is discussed.     

mRNA翻译起始区二级结构优化提高(R)-羰基还原酶的表达及催化效率

为了提高近平滑假丝酵母(Candida parapsilosis CCTCC M203011)的(R)-羰基还原酶在大肠杆菌中的表达水平及催化效率,对酶编码基因mRNA翻译起始区中+1～+78区进行二级结构的优化,并构建了相应的突变体。优化后mRNA翻译起始区的发夹结构明显减少,自由能显著下降(由原始的?9.5kcal/mol降至?5.0kcal/mol),使酶蛋白的表达水平及粗酶比活力分别比优化前提高了4～5倍和61.9%。在高底物浓度(5.0g/L2-羟基苯乙酮)下,优化突变株不对称转化效率较高,产物(R)-苯基乙二醇的光学纯度和产率分别为93.1%e.e.和81.8%,比优化前提高了27.5%和40.5%。研究结果表明:优化mRNA翻译起始区的二级结构,克服蛋白翻译启动的空间位阻,不仅能促进翻译的顺利进行,使目标蛋白得到高效表达,而且有利于蛋白空间结构的正确折叠,有效提高酶蛋白活力及生物催化功能。     

Deciphering the rules by which dynamics of mRNA secondary structure affect translation efficiency in Saccharomyces cerevisiae

Messenger RNA (mRNA) secondary structure decreases the elongation rate, as ribosomes must unwind every structure they encounter during translation. Therefore, the strength of mRNA secondary structure is assumed to be reduced in highly translated mRNAs. However, previous studies in vitro reported a positive correlation between mRNA folding strength and protein abundance. The counterintuitive finding suggests that mRNA secondary structure affects translation efficiency in an undetermined manner. Here, we analyzed the folding behavior of mRNA during translation and its effect on translation efficiency. We simulated translation process based on a novel computational model, taking into account the interactions among ribosomes, codon usage and mRNA secondary structures. We showed that mRNA secondary structure shortens ribosomal distance through the dynamics of folding strength. Notably, when adjacent ribosomes are close, mRNA secondary structures between them disappear, and codon usage determines the elongation rate. More importantly, our results showed that the combined effect of mRNA secondary structure and codon usage in highly translated mRNAs causes a short ribosomal distance in structural regions, which in turn eliminates the structures during translation, leading to a high elongation rate. Together, these findings reveal how the dynamics of mRNA secondary structure coupling with codon usage affect translation efficiency.     

Influence of 5'-terminal cap structure on the initiation of translation of vaccinia virus mRNA

The ability of methylated vaccinia virus mRNA to bind to ribosomes derived from wheat germ of rabbit reticulocyte lysates has been studied after beta elimination, to remove the 5'-terminal m7G, and after "recapping" of beta-eliminated mRNA molecules using guanylyltransferase.guanine-7-methyltransferase complex from vaccinia virions. Removal of m7G from the mRNA results in significant loss of ability to bind to ribosomes and to simulate protein synthesis in vitro. Readdition of m7G, but not of unmethylated guanosine to the 5' end results in recovery of both of these functions. To evaluate the role of 2'-O-methylation of the penultimate ribonucleoside, mRNAs containing m7G-(5')pppA- and m7G(5')pppG- as well as m7G(5')pppAm- and m7G(5')pppGm- ends were synthesized in vitro at limiting S-adenosylmethionine concentrations by vaccinia virus cores. By comparing the cap sequences of ribosome-bound and unbound mRNAs, we concluded that 2'-O-methylation has at most a minor effect compared to that of m7G upon ribosome binding under in vitro conditions. Only at high input mRNA concentrations, at which competition might occur, was there some ribodomal enrichment of mRNAs containing a specific terminal structure, namely m7G(5')pppAm-.