The ribosome must unwind the message before it can read it

The synthesis of a protein requires that the ribosome unwinds structures at the 5'-end of the messenger RNA to prevent inhibition of the translation initiation. Ribosomal protein S1 reversibly binds single-stranded RNA in steps of two or three nucleotides to uncover the site for ribosome binding and subsequent translation.

HFSP Program Grant holders Ignacio Tinoco and Harry Noller and colleagues
authored on Fri, 07 December 2012

Single-molecule studies of translation have revealed that the ribosome pauses at each codon as the new amino acid is introduced and the peptide bond is formed, then quickly—in milliseconds—translocates to the next codon. Structure in the coding region of the messenger RNA (mRNA) must be removed by the powerful helicase activity of the ribosome to allow translocation to proceed. Structure near the initiation site for binding of the small subunit of the ribosome is removed by ribosomal protein S1. The structures in the mRNA and their interactions with the ribosome are part of the closely-controlled regulation of translation and modulation of protein expression.

The step-by-step binding of protein S1 to double-stranded RNA was followed using laser tweezers. The ends of a 274-basepair hairpin RNA (a stem loop) were attached to micron-sized beads. One bead was on a micropipette, the other in the laser trap allowing the the distance between the ends of the RNA to be measured in nanometers, and force applied—in piconewtons (pN).  The S1 protein cannot bind the hairpin RNA if no force is applied to the ends of the hairpin; but as the force is increased the hairpin is destabilized and the S1 binds. At forces above 19 pN, S1 binds and unfolds the hairpin; at forces below 17 pN, S1 dissociates and the hairpin rewinds. We measured the kinetics of binding and dissociation as a function of S1 concentration and force. The rates of binding and dissociation (in the range of 100 s-1) are highly force dependent. The binding rates are linear in S1 concentration; the dissociation rates (off-rates) are independent of S1 concentration, as expected. The single-molecule experiments show step-pause-step trajectories with step size of about 10 nucleotides (nt), see figure. The pauses depend on the base sequence; high GC content causes slower S1 binding and hairpin unwinding;  low GC causes faster S1 dissociation and hairpin re-zipping.

Figure:  The RNA hairpin attached to beads held between the laser trap and a micropipette is shown in the presence of free and bound S1 molecules. On the right are shown: (1) A trajectory during binding of the S1 and unwinding of the RNA, and (2) A trajectory during dissociation of the S1 and re-zipping of the RNA.

Determination of the rate-limiting substeps in the kinetics showed that the rate-limiting substep for unwinding the hairpin was 5 nt; the rate-limiting substep for re-zipping was 2.2 nt. As the substeps are smaller than the 10 nt S1 binding site, the binding must be a multistep process. Protein S1 has 6 oligonucleotide binding domains (2 for ribosome binding, and 4 for mRNA binding) connected by flexible linkers, so it is reasonable for the individual domains to bind independently or cooperatively to produce the observed substeps.

Cellular mRNAs form many small hairpins, which are much less stable than the large hairpin used in this study. Thus the multistep binding of S1 facilitates mRNA unwinding without the need of applying external force. The S1 protein can partially bind to a few nucleotides next to a double-stranded hairpin and wait for a 'breathing fluctuation' of the hairpin to release more nucleotides to allow complete binding. Multistep binding of naturally occurring small hairpins by S1 provides delicate control of translation initiation.


Ribosomal protein S1 unwinds double-stranded RNA in multiple-steps. X. Qu, L. Lancaster, H. F. Noller, C. Bustamante, I. Tinoco, Jr., Proc. Natl. Acad. Sci. USA 109, 14458-63 (2012).

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