70S-scanning initiation is a novel and frequent initiation mode of ribosomal translation in bacteria

Abstract

According to the standard model of bacterial translation initiation, the small ribosomal 30S subunit binds to the initiation site of an mRNA with the help of three initiation factors (IF1-IF3). Here, we describe a novel type of initiation termed "70S-scanning initiation," where the 70S ribosome does not necessarily dissociate after translation of a cistron, but rather scans to the initiation site of the downstream cistron. We detailed the mechanism of 70S-scanning initiation by designing unique monocistronic and polycistronic mRNAs harboring translation reporters, and by reconstituting systems to characterize each distinct mode of initiation. Results show that 70S scanning is triggered by fMet-tRNA and does not require energy; the Shine-Dalgarno sequence is an essential recognition element of the initiation site. IF1 and IF3 requirements for the various initiation modes were assessed by the formation of productive initiation complexes leading to synthesis of active proteins. IF3 is essential and IF1 is highly stimulating for the 70S-scanning mode. The task of IF1 appears to be the prevention of untimely interference by ternary aminoacyl (aa)-tRNA•elongation factor thermo unstable (EF-Tu)•GTP complexes. Evidence indicates that at least 50% of bacterial initiation events use the 70S-scanning mode, underscoring the relative importance of this translation initiation mechanism.protein synthesis | ribosomal functions | translational initiation | 30S-binding initiation | 70S-scanning initiation I t is textbook knowledge that 30S subunits initiate protein synthesis in bacteria; they recognize the initiation site of the mRNA composed of the Shine-Dalgarno (SD) sequence, the AUG codon, and fMet-tRNA, together with three initiation factors (IFs) forming the 30S initiation complex (30SIC). Association of the large 50S subunit triggers the release of the IFs, leading to the 70S initiation complex (70SIC) that enters the elongation phase of translation (reviewed in 1). We term this initiation path the "30S-binding mode" of bacterial initiation. After elongation and termination, it is thought that the ribosome dissociates into its subunits, thus providing 30S subunits for the next round of initiation.The functional role of IF2 is well defined. It can bind directly to the 30S, providing a docking site for fMet-tRNA (2), but it can also enter the 30S subunit as ternary complex fMet-tRNA•IF2•GTP (3). Both IF2 and IF3 are essential for viability. IF3 has a binding site at the 30S interface (4), which explains its antiassociation effect (5, 6), as well as its role in dissociation of the terminating 70S ribosome (7). However, the in vivo concentration of IF3 is 100-fold less (8) than required for full dissociation of 70S in vitro (4). Evidence for the presence of IF3 on 70S ribosomes was reported (9), indicating that the functional spectrum of IF3 is possibly not restricted to an antiassociation effect. Both IF3 and IF2 are also responsible for the fidelity of decoding the initiation AUG by fMet-tRNA Met f at the P site of 30S subun...

Novel Translation Initiation Regulation Mechanism in Escherichia coli ptrB Mediated by a 5′-Terminal AUG

“…Instead, uORF translation appears to be repressed (data not shown) to allow ptrB to take advantage of the 5=-terminal AUG, which strongly binds ribosomes without the added energy requirements of uORF translation. Therefore, the ptrB CDS is regulated independently of uORF translation, which is distinct from previously studied uORF regulation mechanisms (16)(17)(18)(19)(20)(21)(22). Binding of the ribosome to the 5=-uAUG without initiation of translation is also supported by the presence of a tRNA-independent ribosome binding signal in the ptrB toeprints (Fig.…”

“…Energy-independent 70S scanning initiation has been demonstrated recently, with an emphasis on the requirement of an SD sequence for proper positioning (18). In the case of ptrB, the 5=-uAUG may act as a standby-like site for initial ribosomal loading, after which the ribosome can then scan down the mRNA using additional sequences for alignment to compensate for the weak SD sequence.…”

“…In prokaryotes, uORFs exert their effects primarily through translational coupling (14)(15)(16). These effects occur after the ribosome completes translation of the uORF and, rather than dissociating, remains bound to the mRNA and repositions to the downstream initiation region via a scanning-like movement (17,18). uORFs can also encode nascent peptides that cause ribosomal stalling by binding to regions of the peptidyltransferase center, blocking the ribosome exit tunnel, or through attenuation (19,20).…”

Bacillus subtilis, the model Gram‐positive bacterium: 20 years of annotation refinement

“…Another feature of the process of translation has also been revived with the identification of an important role of formylation of the methionine residue loaded on initiator tRNA (Cai et al ., 2017). This revives an open question about the apparent redundant role of formylation in translation previously explored in E. coli where an allosteric modulation of the 70S ribosome structure may shift back to initiation in polycistronic operons without a requirement for the dissociation of ribosomal subunits during the translation of contiguous cistrons (Petersen et al ., 1976; Yamamoto et al ., 2016). …”

Noncanonical Translation Initiation Comes of Age

“…An essential difference between initiation on leadered versus leaderless mRNAs is that the latter begins with the binding of a 70S ribosome to the 5= end. While the 30S mode of initiation has long been considered dominant, a recent proposal suggested that initiation by scanning 70S ribosomes is the frequent mode of translation of polycistronic mRNAs (15).…”