Precise tuning of bacterial translation initiation by non-equilibrium 5’-UTR unfolding observed in single mRNAs

Noncoding, structured 5’- untranslated regions (5’-UTRs) of messenger RNAs (mRNAs) control translation efficiency by forming structures that can either recruit or repel the ribosome. Here we exploit a bacterial, preQ1-sensing translational riboswitch to probe how binding of a small ligand controls binding of the bacterial ribosome to the Shine-Dalgarno (SD) sequence. Combining single-molecule fluorescence microscopy with mutational analyses, we find that the stability of 30S ribosomal subunit binding is inversely correlated with the free energy needed to unfold the 5’-UTR during mRNA accommodation from the standby site to the binding cleft. Ligand binding stabilizes 5’-UTR structure to both antagonize 30S recruitment and accelerate 30S dissociation. Depletion of small ribosomal subunit protein S1, known to resolve structured 5’-UTRs, further increases the energetic penalty for mRNA accommodation. The resulting model of rapid standby site exploration followed by gated non-equilibrium unfolding of the 5’-UTR during accommodation provides a mechanistic understanding of translation efficiency.

Ribosome profiling in Mycobacterium tuberculosis reveals robust leaderless translation

Mycobacterium tuberculosis, which causes tuberculosis, expresses a large proportion of leaderless transcripts lacking the canonical bacterial translation initiation signals. The role leaderless genes play in the physiology of this pathogen, which can undergo prolonged periods of non-replicating persistence in the host, is currently unknown. We have previously demonstrated that levels of leaderless transcription increase under conditions of nutrient starvation. However, little is known about the implications of this for persistent infection. Here, we performed ribosome profiling to characterise the translational landscape of M. tuberculosis in vitro. Our data reveals robust leaderless translation in the pathogen and points towards different mechanisms for their initiation of translation compared to canonical Shine-Dalgarno genes. Furthermore, under conditions of nutrient starvation, we found a significant global up-regulation of leaderless genes in the translatome. Our data represents a rich resource for others seeking to understand translational regulation not only in M. tuberculosis but in bacterial pathogens.

Real-time structural dynamics of late steps in bacterial translation initiation visualized using time-resolved cryogenic electron microscopy

Bacterial translation initiation entails the tightly regulated joining of the 50S ribosomal subunit to an initiator transfer RNA (fMet-tRNAfMet)-containing 30S ribosomal initiation complex (IC) to form a 70S IC that subsequently matures into a 70S elongation-competent complex (70S EC). Rapid and accurate 70S IC formation is promoted by 30S IC-bound initiation factor (IF) 1 and the guanosine triphosphatase (GTPase) IF2, both of which must ultimately dissociate from the 70S IC before the resulting 70S EC can begin translation elongation1. Although comparison of 30S2–6 and 70S5,7–9 IC structures have revealed that the ribosome, IFs, and fMet-tRNAfMet can acquire different conformations in these complexes, the timing of conformational changes during 70S IC formation, structures of any intermediates formed during these rearrangements, and contributions that these dynamics might make to the mechanism and regulation of initiation remain unknown. Moreover, lack of an authentic 70S EC structure has precluded an understanding of ribosome, IF, and fMet-tRNAfMet rearrangements that occur upon maturation of a 70S IC into a 70S EC. Using time-resolved cryogenic electron microscopy (TR cryo-EM)10 we report the first, near-atomic-resolution view of how a time-ordered series of conformational changes drive and regulate subunit joining, IF dissociation, and fMet-tRNAfMet positioning during 70S EC formation. We have found that, within ~20–80 ms, rearrangements of the 30S subunit and IF2, uniquely captured in its GDP•Pi-bound state, stabilize fMet-tRNAfMet in its intermediate, ‘70S P/I’, configuration7 and trigger dissociation of IF1 from the 70S IC. Within the next several hundreds of ms, dissociation of IF2 from the 70S IC is coupled to further remodeling of the ribosome that positions fMet-tRNAfMet into its final, ‘P/P’, configuration within the 70S EC. Our results demonstrate the power of TR cryo-EM to determine how a time-ordered series of conformational changes contribute to the mechanism and regulation of one of the most fundamental processes in biology.

A Unique Ribosome Signature Reveals Bacterial Translation Initiation Sites

ABSTRACTWhile methods for annotation of genes are increasingly reliable the exact identification of the translation initiation site remains a challenging problem. Since the N-termini of proteins often contain regulatory and targeting information developing a robust method for start site identification is crucial. Ribosome profiling reads show distinct patterns of read length distributions around translation initiation sites. These patterns are typically lost in standard ribosome profiling analysis pipelines, when reads from footprints are adjusted to determine the specific codon being translated. Using these unique signatures we build a model capable of predicting translation initiation sites and demonstrate its high accuracy using N-terminal proteomics. Applying this to prokaryotic samples, we re-annotate translation initiation sites and provide evidence of N-terminal truncations and elongations of annotated coding sequences. These re-annotations are supported by the presence of Shine-Dalgarno sequences, structural and sequence based features and N-terminal peptides. Finally, our model identifies 61 novel genes previously undiscovered in the genome.