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.     

Molecular basis of ribosome recognition and mRNA hydrolysis by the E. coli YafQ toxin

Bacterial type II toxin-antitoxin modules are protein–protein complexes whose functions are finely tuned by rapidly changing environmental conditions. E. coli toxin YafQ is suppressed under steady state growth conditions by virtue of its interaction with its cognate antitoxin, DinJ. During stress, DinJ is proteolytically degraded and free YafQ halts translation by degrading ribosome-bound mRNA to slow growth until the stress has passed. Although structures of the ribosome with toxins RelE and YoeB have been solved, it is unclear what residues among ribosome-dependent toxins are essential for mediating both recognition of the ribosome and the mRNA substrate given their low sequence identities. Here we show that YafQ coordinates binding to the 70S ribosome via three surface-exposed patches of basic residues that we propose directly interact with 16S rRNA. We demonstrate that YafQ residues H50, H63, D67 and H87 participate in acid-base catalysis during mRNA hydrolysis and further show that H50 and H63 functionally complement as general bases to initiate the phosphodiester cleavage reaction. Moreover YafQ residue F91 likely plays an important role in mRNA positioning. In summary, our findings demonstrate the plasticity of ribosome-dependent toxin active site residues and further our understanding of which toxin residues are important for function.     

Expression of tetanus toxin fragment C in E. coli: high level expression by removing rare codons.

Tetanus toxin fragment C had been previously expressed in Escherichia coli at 3-4% cell protein. The codon bias for tetanus toxin in Clostridium tetani is very different from that of highly expressed homologous genes in E. coli, resulting in the presence of many rare E. coli codons in the sequence encoding fragment C. We have replaced the coding sequence by sequence optimized for codon usage in E. coli, and show that the expression of fragment C is increased. Although the level of mRNA also increased this appeared to be a secondary consequence of more efficient translation. Complete sequence replacement increased expression to approximately 11-14% cell protein but only after the promoter strength had been improved.     

Growth and translation inhibition through sequence-specific RNA binding by Mycobacterium tuberculosis VapC toxin

The Mycobacterium tuberculosis genome harbors an unusually large number of toxin-antitoxin (TA) modules. Curiously, over half of these are VapBC (virulence-associated protein) family members. Nonetheless, the cellular target, precise mode of action, and physiological role of the VapC toxins in this important pathogen remain unclear. To better understand the function of this toxin family, we studied the features and biochemical properties of a prototype M. tuberculosis VapBC TA system, vapBC-mt4 (Rv0596c-Rv0595c). VapC-mt4 expression resulted in growth arrest, a hallmark of all TA toxins, in Escherichia coli, Mycobacterium smegmatis, and M. tuberculosis. Its expression led to translation inhibition accompanied by a gradual decrease in the steady-state levels of several mRNAs. VapC-mt4 exhibited sequence-specific endoribonuclease activity on mRNA templates at ACGC and AC(A/U)GC sequences. However, the cleavage activity of VapC-mt4 was comparatively weak relative to the TA toxin MazF-mt1 (Rv2801c). Unlike other TA toxins, translation inhibition and growth arrest preceded mRNA cleavage, suggesting that the RNA binding property of VapC-mt4, not RNA cleavage, initiates toxicity. In support of this hypothesis, expression of VapC-mt4 led to an increase in the recovery of total RNA with time in contrast to TA toxins that inhibit translation via direct mRNA cleavage. Additionally, VapC-mt4 exhibited stable, sequence-specific RNA binding in an electrophoretic mobility shift assay. Finally, VapC-mt4 inhibited protein synthesis in a cell-free system without cleaving the corresponding mRNA. Therefore, the activity of VapC-mt4 is mechanistically distinct from other TA toxins because it appears to primarily inhibit translation through selective, stable binding to RNA.     

Chemical transformation of eremofortin C into PR toxin

The natural products of both eremofortin C (EC) and PR toxin are secondary metabolites of Penicillium roqueforti. Because the chemical structures of EC and PR toxin are closely related to each other and differ only by a hydroxyl functional group in EC and an aldehyde functional group in PR toxin at the C-12 position, the chemical transformation of EC into PR toxin was investigated. Oxidation with a chromic anhydride-pyridine complex was found to be the most satisfactory method.     

Chemical transformation of eremofortin C into PR toxin.

The natural products of both eremofortin C (EC) and PR toxin are secondary metabolites of Penicillium roqueforti. Because the chemical structures of EC and PR toxin are closely related to each other and differ only by a hydroxyl functional group in EC and an aldehyde functional group in PR toxin at the C-12 position, the chemical transformation of EC into PR toxin was investigated. Oxidation with a chromic anhydride-pyridine complex was found to be the most satisfactory method.     

Secondary metabolites resulting from degradation of PR toxin by Penicillium roqueforti.

PR toxin is a secondary metabolite of the fungus Penicillium roqueforti. It is lethal to rats, mice, and cats. Usually, the amount of PR toxin in the culture medium decreases from its maximum on day 15 to zero within 3 to 4 days. We found that two were secondary metabolites produced in the culture medium of this fungus while the production of PR toxin was decreasing. We isolated and purified the two compounds in pure and colorless crystalline form. On the basis of elemental analysis and mass, 1H and 13C nuclear magnetic resonance, infrared, and UV spectroscopies, the two compounds were identified as PR-imine (C17H21O5N) and PR-amide (C17H21O6N). The structures of both compounds and of PR toxin (C17H20O6) were closely related, and the peak production of PR toxin appeared earlier than those of PR-imine and PR-amide. Moreover, PR toxin was transformed to PR-imine when PR toxin was incubated with the culture medium on a given culture day. Thus, we propose that PR toxin is degraded into PR-imine and PR-amide in the culture medium of P. roqueforti.     

翻译起始区二级结构对荧光素酶基因在COS-7细胞中表达效率的影响

在荧光素酶基因起始密码子ATG下游插入4种串连重复的密码子(6×ATT ,3×ATT ,6×GCC与3×GCC) ,得到具有不同二级结构的翻译起始区(translationinitiationregion ,TIR) ,以研究TIR二级结构对该基因在COS 7细胞中表达的影响.Northern印迹结果显示,4种重组子mRNA的转录水平没有显著差异,而Western印迹与酶活性检测表明,与野生型结构相比,6×ATT与3×ATT能显著提高荧光素酶的表达量与活性,而6×GCC结构的表达量明显下降.使用计算机辅助分析软件,扫描上述TIR结构发现,TIR稳定性或二级结构复杂度是导致上述表达差异的主要原因.     

tRNA is a new target for cleavage by a MazF toxin

Toxin-antitoxin (TA) systems play key roles in bacterial persistence, biofilm formation and stress responses. The MazF toxin from the Escherichia coli mazEF TA system is a sequence- and single-strand-specific endoribonuclease, and many studies have led to the proposal that MazF family members exclusively target mRNA. However, recent data indicate some MazF toxins can cleave specific sites within rRNA in concert with mRNA. In this report, we identified the repertoire of RNAs cleaved by Mycobacterium tuberculosis toxin MazF-mt9 using an RNA-seq-based approach. This analysis revealed that two tRNAs were the principal targets of MazF-mt9, and each was cleaved at a single site in either the tRNA^Pro14 D-loop or within the tRNA^Lys43 anticodon. This highly selective target discrimination occurs through recognition of not only sequence but also structural determinants. Thus, MazF-mt9 represents the only MazF family member known to target tRNA and to require RNA structure for recognition and cleavage. Interestingly, the tRNase activity of MazF-mt9 mirrors basic features of eukaryotic tRNases that also generate stable tRNA-derived fragments that can inhibit translation in response to stress. Our data also suggest a role for tRNA distinct from its canonical adapter function in translation, as cleavage of tRNAs by MazF-mt9 downregulates bacterial growth.     

The dependence of the E.coli gene expression level on the structure of the translation initiation region (TIR). IV. Distal complementary TIR interactions with the mRNA coding region

To evaluate the effect on translation of distal regions of the encoding mRNA part capable of the complementary binding to the ribosome binding site (RBS), a series of plasmids were constructed containing fragments inserted into the il3 gene and determining secondary interactions in mRNA. A comparison of the levels of the in vivo gene expression showed that the complementary interactions of the translation initiation region (TIR) with distal regions of the mRNA encoding part affect translation. The effectiveness of these interactions decreased with an increase in the distance between the RBS and the complementary mRNA region, whereas the secondary structure formed by the TIR and the adjacent mRNA region was more stable despite the presence of regions in mRNA capable of forming energetically more favorable structures involving these elements.     

Conformational change in the catalytic site of the ribonuclease YoeB toxin by YefM antitoxin

The eubacterial chromosome encodes various addiction modules that control global levels of translation through RNA degradation. Crystal structures of the Escherichia coli YefM2 (antitoxin)-YoeB (toxin) complex and the free YoeB toxin have been determined. The structure of the heterotrimeric complex reveals an asymmetric disorder-to-order recognition strategy, in which one C terminus of the YefM homodimer exclusively interacts with an atypical microbial ribonuclease (RNase) fold of YoeB. Comparison with the YefM-free YoeB structure indicates a conformational rearrangement of the RNase catalytic site of YoeB, induced by interaction with YefM. Complementary biochemical experiments demonstrate that the YoeB toxin has an in vitro RNase activity that preferentially cleaves at the 3' end of purine ribonucleotides.     

mRNA helicases: the tacticians of translational control

The translation initiation step in eukaryotes is highly regulated and rate-limiting. During this process, the 40S ribosomal subunit is usually recruited to the 5' terminus of the mRNA. It then migrates towards the initiation codon, where it is joined by the 60S ribosomal subunit to form the 80S initiation complex. Secondary structures in the 5' untranslated region (UTR) can impede binding and movement of the 40S ribosome. The canonical eukaryotic translation initiation factor eIF4A (also known as DDX2), together with its accessory proteins eIF4B and eIF4H, is thought to act as a helicase that unwinds secondary structures in the mRNA 5' UTR. Growing evidence suggests that other helicases are also important for translation initiation and may promote the scanning processivity of the 40S subunit, synergize with eIF4A to 'melt' secondary structures or facilitate translation of a subset of mRNAs.     

Codon usage determines translation rate in Escherichia coli

We wish to determine whether differences in translation rate are correlated with differences in codon usage or with differences in mRNA secondary structure. We therefore inserted a small DNA fragment in the lacZ gene either directly or flanked by a few frame-shifting bases, leaving the reading frame of the lacZ gene unchanged. The fragment was chosen to have "infrequent" codons in one reading frame and "common" codons in the other. The insert in these constructs does not seem to give mRNAs that are able to form extensive secondary structures. The translation time for these modified lacZ mRNAs was measured with a reproducibility better than plus or minus one second. We found that the mRNA with infrequent codons inserted has an approximately three-seconds longer translation time than the one with common codons. In another set of experiments we constructed two almost identical lacZ genes in which the lacZ mRNAs have the potential to generate stem structures with stabilities of about -75 kcal/mol. In this way we could investigate the influence of mRNA structure on translation rate. This type of modified gene was generated in two reading frames with either common or infrequent codons similar to our first experiments. We find that the yield of protein from these mRNAs is reduced, probably due to the action in vivo of an RNase. Nevertheless, the data do not indicate that there is any effect of mRNA secondary structure on translation rate. In contrast, our data persuade us that there is a difference in translation rate between infrequent codons and common codons that is of the order of sixfold.