ΔGunfold leaderless, a package for high-throughput analysis of translation initiation regions (TIRs) at the transcriptome scale and for leaderless mRNA optimization

Translation initiation is an essential step for fidelity of gene expression, in which the ribosome must bind to the translation initiation region (TIR) and position the initiator tRNA in the P-site (1). For this to occur correctly, the TIR encompassing the ribosome binding site (RBS) needs to be highly accessible (2-5). ΔGunfold is a metric for computing accessibility of the TIR, but there is no automated way to compute it manually with existing software/tools limiting throughput. ΔGunfold leaderless allows users to automate the ΔGunfold calculation to perform high-throughput analysis. Importantly, ΔGunfold leaderless allows calculation of TIRs of both leadered mRNAs and leaderless mRNAs which lack a 5' UTR and which are abundant in bacterial, archaeal, and mitochondrial transcriptomes (4, 6, 7). The ability to analyze leaderless mRNAs also allows one additional feature where users can computationally optimize leaderless mRNA TIRs to maximize their gene expression (8, 9). The ΔGunfold leaderless package facilitates high-throughput calculations of TIR accessibility, is designed to calculate TIR accessibility for leadered and leaderless mRNA TIRs which are abundant in bacterial/archaeal/organellar transcriptomes and allows optimization of leaderless mRNA TIRs for biotechnology.

Enhanced translation of leaderless mRNAs under oxidative stress in Escherichia coli

The bacterial response to oxidative stress requires the adaptation of the proteome to the hostile environment. It has been reported that oxidative stress induces a strong and global inhibition of both, transcription and translation. Nevertheless, whereas it is well known that transcription of a small group of genes is induced thanks to transcription factors such as OxyR and SoxR, an equivalent mechanism has not been described for translation. Here we report that whereas canonical translation that depends on Shine Dalgarno recognition is inhibited by oxidative stress in Escherichia coli, the translation of leaderless mRNA (lmRNA) is enhanced under such conditions. Both, inhibition of canonical translation and enhancement of lmRNA translation, depend on the production of (p)ppGpp. We propose that such a mechanism would allow bacteria to rapidly adapt their proteome to hostile conditions and is, perhaps, a general strategy to confront strong stressful conditions.

A combination of mRNA features influence the efficiency of leaderless mRNA translation initiation

Bacterial translation is thought to initiate by base pairing of the 16S rRNA and the Shine–Dalgarno sequence in the mRNA’s 5′ untranslated region (UTR). However, transcriptomics has revealed that leaderless mRNAs, which completely lack any 5′ UTR, are broadly distributed across bacteria and can initiate translation in the absence of the Shine–Dalgarno sequence. To investigate the mechanism of leaderless mRNA translation initiation, synthetic in vivo translation reporters were designed that systematically tested the effects of start codon accessibility, leader length, and start codon identity on leaderless mRNA translation initiation. Using these data, a simple computational model was built based on the combinatorial relationship of these mRNA features that can accurately classify leaderless mRNAs and predict the translation initiation efficiency of leaderless mRNAs. Thus, start codon accessibility, leader length, and start codon identity combine to define leaderless mRNA translation initiation in bacteria.

The Universally Conserved ATPase YchF Regulates Translation of Leaderless mRNA in Response to Stress Conditions

The universally conserved P-loop GTPases control diverse cellular processes, like signal transduction, ribosome assembly, cell motility, and intracellular transport and translation. YchF belongs to the Obg-family of P-loop GTPases and is one of the least characterized member of this family. It is unique because it preferentially hydrolyses ATP rather than GTP, but its physiological role is largely unknown. Studies in different organisms including humans suggest a possible role of YchF in regulating the cellular adaptation to stress conditions. In the current study, we explored the role of YchF in the model organism Escherichia coli. By western blot and promoter fusion experiments, we demonstrate that YchF levels decrease during stress conditions or when cells enter stationary phase. The decline in YchF levels trigger increased stress resistance and cells lacking YchF are resistant to multiple stress conditions, like oxidative stress, replication stress, or translational stress. By in vivo site directed cross-linking we demonstrate that YchF interacts with the translation initiation factor 3 (IF3) and with multiple ribosomal proteins at the surface of the small ribosomal subunit. The absence of YchF enhances the anti-association activity of IF3, stimulates the translation of leaderless mRNAs, and increases the resistance against the endoribonuclease MazF, which generates leaderless mRNAs during stress conditions. In summary, our data identify YchF as a stress-responsive regulator of leaderless mRNA translation.

Reconstitution of mammalian mitochondrial translation system capable of correct initiation and long polypeptide synthesis from leaderless mRNA

Mammalian mitochondria have their own dedicated protein synthesis system, which produces 13 essential subunits of the oxidative phosphorylation complexes. We have reconstituted an in vitro translation system from mammalian mitochondria, utilizing purified recombinant mitochondrial translation factors, 55S ribosomes from pig liver mitochondria, and a tRNA mixture from either Escherichia coli or yeast. The system is capable of translating leaderless mRNAs encoding model proteins (DHFR and nanoLuciferase) or some mtDNA-encoded proteins. We show that a leaderless mRNA, encoding nanoLuciferase, is faithfully initiated without the need for any auxiliary factors other than IF-2mt and IF-3mt. We found that the ribosome-dependent GTPase activities of both the translocase EF-G1mt and the recycling factor EF-G2mt are insensitive to fusidic acid (FA), the translation inhibitor that targets bacterial EF-G homologs, and consequently the system is resistant to FA. Moreover, we demonstrate that a polyproline sequence in the protein causes 55S mitochondrial ribosome stalling, yielding ribosome nascent chain complexes. Analyses of the effects of the Mg concentration on the polyproline-mediated ribosome stalling suggested the unique regulation of peptide elongation by the mitoribosome. This system will be useful for analyzing the mechanism of translation initiation, and the interactions between the nascent peptide chain and the mitochondrial ribosome.

A combinatorial model predicts leaderless mRNA start codon selection in C. crescentus

Bacterial translation is thought to initiate by base-pairing of the 16S rRNA and the Shine-Dalgarno sequence in the mRNA’s 5’ UTR. However, transcriptomics has revealed that leaderless mRNAs, which completely lack any 5’ UTR, are broadly distributed across bacteria and can initiate translation in the absence of the Shine-Dalgarno sequence. To investigate the mechanism of leaderless mRNA translation initiation, synthetic in vivo translation reporters were designed that systematically tested the effects of start codon accessibility, leader length, and start codon identity on leaderless mRNA translation initiation. Using this data, a simple computational model was built based on the combinatorial relationship of these mRNA features which can accurately classify leaderless mRNAs and predict the translation initiation efficiency of leaderless mRNAs. Thus, start codon accessibility, leader length, and start codon identity combine to define leaderless mRNA translation initiation in bacteria.

One Gene and Two Proteins: a Leaderless mRNA Supports the Translation of a Shorter Form of theShigellaVirF Regulator

ABSTRACTVirF, an AraC-like activator, is required to trigger a regulatory cascade that initiates the invasive program ofShigellaspp., the etiological agents of bacillary dysentery in humans. VirF expression is activated upon entry into the host and depends on many environmental signals. Here, we show that thevirFmRNA is translated into two proteins, the major form, VirF30(30 kDa), and the shorter VirF21(21 kDa), lacking the N-terminal segment. By site-specific mutagenesis and toeprint analysis, we identified the translation start sites of VirF30and VirF21and showed that the two different forms of VirF arise from differential translation. Interestingly,in vitroandin vivotranslation experiments showed that VirF21is also translated from a leaderless mRNA (llmRNA) whose 5′ end is at position +309/+310, only 1 or 2 nucleotides upstream of the ATG84 start codon of VirF21. The llmRNA is transcribed from a gene-internal promoter, which we identified here. Functional analysis revealed that while VirF30is responsible for activation of the virulence system, VirF21negatively autoregulatesvirFexpression itself. Since VirF21modulates the intracellular VirF levels, this suggests that transcription of the llmRNA might occur when the onset of the virulence program is not required. We speculate that environmental cues, like stress conditions, may promote changes invirFmRNA transcription and preferential translation of llmRNA.IMPORTANCEShigellaspp. are a major cause of dysentery in humans. In bacteria of this genus, the activation of the invasive program involves a multitude of signals that act on all layers of the gene regulatory hierarchy. By controlling the essential genes for host cell invasion, VirF is the key regulator of the switch from the noninvasive to the invasive phenotype. Here, we show that theShigella virFgene encodes two proteins of different sizes, VirF30and VirF21, that are functionally distinct. The major form, VirF30, activates the genes necessary for virulence, whereas the minor VirF21, which shares the C-terminal two-thirds of VirF30, negatively autoregulatesvirFexpression itself. VirF21is transcribed from a newly identified gene-internal promoter and, moreover, is translated from an unusual leaderless mRNA. The identification of a new player in regulation adds complexity to the regulation of theShigellainvasive process and may help development of new therapies for shigellosis.