Translation initiation site of mRNA is selected through dynamic interaction with the ribosome

Fig. 1.. smFRET experimental setup. (A) Design of mRNA constructs. Left, mRNA was labeled with Cy3 and Cy5 dyes through complementary DNA handles at the 3′ and 5′ ends, respectively. The 5′ handle also contained a biotin tag for immobilization onto the slide surface. Middle, The mRNA sequences (5′ to 3′) between the two handles are shown. Sequences identical to F+18 are denoted as dashes for clarity. The approximate ribosome footprint is indicated by the purple bar. Right, the secondary structure of the hairpin (hp). (B) Schematic of a typical experimental procedure. Left, The 30S subunits and indicated components were incubated with immobilized, dye pair–labeled mRNA. FRET time traces were recorded and used to construct the ensemble EFRET distribution (gray histogram), which usually could be fit to a Gaussian function (green curve). Right, after recording, buffer was injected to remove unbound components. FRET time traces were recorded again, and the ensemble EFRET was constructed (orange line); PEG, polyethylene glycol.

Fig. 2.. Accommodation of unstructured mRNA on 30S subunits is facilitated by initiator tRNA and initiation factors. The F+18 construct was used to measure the conformational changes of unstructured mRNA at various ribosome-binding states. (A) mRNA alone. (B) 70S IC formed in the presence of IF1, IF2, and IF3. (C) 30S IC. (D) 30S PIC. EFRET distributions before (gray histograms) and after (orange lines) buffer washing and a representative FRET time trace are shown. (E) Hidden Markov modeling for the 30S PIC. A FRET time trace (20 Hz) was fit to three EFRET states, centered at around 0.1, 0.3, and 0.8, and referred to as the low-, middle-, and high-FRET states, respectively. (F) Summary of transition rates from one state (Top) to another (Left) from the hidden Markov modeling analysis. The rates are in s−1; n/d, not detectable. (G) A model to illustrate a possible conformational change of mRNA in the 30S PIC.

Fig. 3.. mRNAs with weak or null SD sequences are compromised in recruiting 30S subunits. (A–D) EFRET histograms of FwSD+18 (with a weak SD sequence AGGA) in the states of mRNA alone (A), 30S PIC (B), 30S PIC+IF1/3 (C), and 30S IC (D). Gray bars and orange lines represent histograms before and after buffer washing, respectively. A representative FRET time trace is shown in B–D. (E–H) Same as in A–D, except that the SD-free construct FnSD+18 was measured. (I) Association (kon) and dissociation (koff) rate constants of 30S PIC with FwSD+18 (wSD) or FnSD+18 (nSD), measured in the presence (+) or absence (−) of both IF1 and IF3. Errors were estimated by a bootstrapping method (SI Appendix, Methods).

Fig. 4.. Binding of initiator tRNA stabilizes the 30S PIC formed with structured mRNA. (A–C) EFRET histograms of F+11hp in the states of mRNA alone (A), 30S PIC (B), and 30S IC (C). Gray bars and orange lines represent histograms before and after buffer washing, respectively. A representative FRET time trace is shown for the 30S PIC. (D–F) Corresponding to A–C, except that F+14 was measured. (G–I) Corresponding to A–C, except that F+11 was measured. (J–L) Corresponding to A–C, except that F+8 was measured.

Fig. 5.. The ribosome can unwind up to 3 bp of the downstream mRNA structure after the binding of initiator tRNA. (A) Experimental setup for optical tweezers. An anti-digoxigenin antibody–coated polystyrene bead (Left) and a streptavidin-coated bead (Right) were attached to the ends of mRNA through the digoxigenin- and biotin-labeled DNA handles, respectively. The mRNA sequences (5′ to 3′) between the two handles are shown. Sequences identical to T+11hp are denoted as dashes for clarity. The approximate ribosome footprint is indicated by the purple bar, which overlaps part of the downstream hairpins (hp, hpM, and hpE). In hpM (red boxed), the mismatched and bulged bases of hp (blue boxed) were changed such that the whole stem was correctly base paired. For hpE, see SI Appendix, Fig. S4A for detail. (B and C) Difference in unfolding force (B) and size (C) of the downstream hairpins. For each of the indicated mRNA constructs, the unfolding force and size of the hairpin in the designated ribosome-binding states (30S PIC, 30S IC, 70S IC, and 30S PIC+IF1/3) were plotted as the difference from that of the mRNA alone. Error bars represent SDs; n/a, not available.

Fig. 6.. Initiation factors facilitate the association of 30S subunits with structured mRNA. (A–E) EFRET histograms of F+11hp in the state of 30S PIC without initiation factors (A), with IF1 (B), with IF3 (C), with both IF1 and IF3 (D), or with IF2 + 1 mM GTP (E). Gray bars and orange lines represent histograms before and after buffer washing, respectively. The blue dashed line is shown as a reference across panels to indicate the major EFRET peak (0.17) of 30S PIC+IF3 (C). (F–J) Corresponding to A–E, except that the state was 30S IC (with initiator tRNA). The purple dashed line is a reference indicating the major EFRET peak (0.11) of 30S IC (F). The same blue dashed line as in A–E is also shown for comparison. (K–O) Corresponding to A–E, except that F+8 was measured. The blue dashed line is a reference indicating the major EFRET peak (0.24) of 30S PIC+IF3 (M). (P–T) Corresponding to K–O, except that the state was 30S IC (with initiator tRNA). The purple dashed line is a reference indicating the major EFRET peak (0.09) of 30S IC (P). The same blue dashed line as in K–O is also shown for comparison.

Fig. 7.. IF3 facilitates the disassembly of 30S IC. (A) Time-dependent disassembly of F+18 and F+11hp in the 30S IC states. Normalized percentages of F+18 and F+11hp retained in the 30S complexes were measured after buffer washing (Buf. wash; black filled circles) or IF3 washing (blue filled circles). Each data set was fitted to a kinetic model containing two rate constants (SI Appendix, Table S3). (B and C) Time-dependent EFRET histograms of F+11hp in the 30S IC state after buffer (B) and IF3 (C) washing. The blue arrow indicates the IF3-dependent emergency of the middle FRET state. (D) Time-dependent disassembly of F+8 in the 30S IC state after buffer washing (black filled circles) or IF3 washing (blue filled circles) and F+8 in the 30S PIC+IF3 state after buffer washing (gray open circle). (E and F) Corresponding to B and C, except that F+8 in the 30S IC state was measured. In A and D, shown are data averaged from three (with error bars) or two (without error bars) independent experiments. The error bars represent SDs.

Fig. 8.. A model for the dynamic RBS selection by the 30S subunit during initiation. The initiation factors (IF1, IF2, and IF3) may bind to the 30S subunit before or after its binding to mRNA. Step 1, association of the 30S subunit to mRNA through SD:anti-SD base pairing. Step 2, movement of the start codon to the proximity of the P site, which is assisted by initiation factors. Step 3, binding of the initiator tRNA to the P site, such that the downstream RBS is placed at around the mRNA entrance site. Step 4, closure of the entrance site if the RBS can be accommodated completely. Step 5, stable 70S IC is formed in the presence of 50S subunits (5a), or the initiator tRNA is dissociated and the reaction is reverted to a previous step (5b). The corresponding FRET states are shown in each step.