Results Accommodation of Unstructured mRNA on 30S Subunits Is Facilitated by Initiator tRNA and Initiation Factors. Thus, initiation factors may accelerate the assembly–disassembly kinetics of 30S ICs such that a correct RBS can be quickly identified by the 30S subunit during initiation. IF3 (and likely IF2 as well) promoted dissociation of the initiator tRNA from the complex, followed by disassembly of the 30S–mRNA complex. However, the formed 30S ICs with structured mRNA were not stable. Finite unwinding of the downstream RBS structure was observed only when initiator tRNA was present. Initiation factors could facilitate further accommodation of the mRNA. We found that mRNA was initially recruited to the 30S subunit through the SD sequence, while the downstream RBS was only dynamically associated, especially when it formed a structure. To explore the accommodation of mRNA with various sequences and structures, we measured the interaction between mRNA and the 30S subunit at the initiation stage using single-molecule Förster resonance energy transfer (smFRET) ( 27) and optical tweezers ( 28). Overall, how mRNA structures affect initiation depends on the structure’s position, stability, and flanking sequences, among others, yet the detailed molecular mechanisms remain elusive. The entrance site possesses helicase activity that can unwind mRNA structures during elongation ( 23– 26), but whether it is also involved in initiation is unknown. The downstream mRNA that enters the 30S subunit is located at position +13 to +15 and is surrounded by ribosomal proteins uS3, uS4, and uS5 ( 2, 23). A moderate mRNA structure containing the SD sequence can interact rapidly with the 30S subunit through a neighboring single strand, but further stabilization of the complex requires the participation of initiation factors and initiator tRNA ( 7). Consistent with this finding, several studies have systematically varied the sequence of the 5′ untranslated region (5′ UTR) ( 21), the hairpin harboring the start codon ( 18), or the synonymous codons of a reporter protein ( 22) and have shown that secondary structures formed near the RBS tend to down-regulate translation. The exposed single-stranded initiation site may facilitate ribosome association. A genome-wide analysis showed that, in polycistronic mRNAs, each open reading frame forms distinct structures, which are separated by less-structured regions centered at the start codon (roughly from positions −25 to +25 the first nucleotide of the start codon is designated as +1) ( 20). Our study provides insights into how the bacterial ribosome identifies a typical translation initiation site from mRNA.ĭuring the initiation process, the 30S subunit may encounter various mRNA structures that are used to regulate translation ( 17– 19). Thus, initiation factors may accelerate the dynamic assembly–disassembly process of 30S–mRNA complexes such that the correct RBS can be preferentially selected. IF3 promotes dissociation by dismissing the bound initiator tRNA. Meanwhile, the formed complex of the 30S subunit with structured mRNA is not stable and tends to disassociate. The initiator transfer RNA (tRNA) further helps the 30S subunit accommodate mRNA and unwind up to 3 base pairs of the RBS structure. The mRNA is either dissociated or stabilized by initiation factors, such as initiation factor 3 (IF3). We found that the 30S subunit initially binds to mRNA through the SD sequence, whereas the downstream RBS undergoes dynamic motions, especially when it forms structures. Here, we used single-molecule techniques to tackle this long-standing issue. How the rather short and degenerate information inside the RBS can be correctly accommodated by the ribosome is not well understood. Both the SD sequence and the start codon comprise the core ribosome-binding site (RBS), to which the 30S ribosomal subunit binds to initiate translation. In bacteria, the start codon is usually preceded by a 4- to 6-mer adenosine/guanosine-rich Shine–Dalgarno (SD) sequence. Initiation of protein synthesis from the correct start codon of messenger RNA (mRNA) is crucial to translation fidelity.
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