Table 1.

Summary of smFRET results

mRNA	Labeling scheme	Fluorescent Labeling Scheme													FRET Pair	FRET Efficiencies			(Eongoing – Ehybrid)/(Eclassical – Ehybrid)
																Stalled-PRE Classic	Stalled-PRE Hybrid	INTongoing
mRNA-6,7	A	AUG	UAU	UAU	UAU	UAU	UUC	GUG	CGU	UAU	UAU	UAU	UAU	UAU	V7(Cy3)-L11(Cy5)	0.73 ± 0.01 (Fig 1C)	0.49 ± 0.02 (Fig 1C)	0.65 ± 0.01 (Fig 1F)	0.67
		fMet	Tyr	Tyr	Tyr	Tyr	Phe	Val	Arg	Tyr	Tyr	Tyr	Tyr	Tyr
		1	2	3	4	5	6	7	8	9	10	11	12	13
	B	AUG	UAU	UAU	UAU	UAU	UUC	GUG	CGU	UAU	UAU	UAU	UAU	UAU	F6(Cy5)-V7(Cy3)	0.68 ± 0.01 (Fig 2C)	0.39 ± 0.01 (Fig 2C)	0.58 ± 0.01 (Fig 2F)	0.66
		fMet	Tyr	Tyr	Tyr	Tyr	Phe	Val	Arg	Tyr	Tyr	Tyr	Tyr	Tyr
		1	2	3	4	5	6	7	8	9	10	11	12	13
	C	AUG	UAU	UAU	UAU	UAU	UUC	GUG	CGU	UAU	UAU	UAU	UAU	UAU	F6(Cy3)-V7(Cy5)	0.73 ± 0.01 (Fig S1C)	0.40 ± 0.01 (Fig S1C)	0.57 ± 0.02 (Fig S1F)	0.52
		fMet	Tyr	Tyr	Tyr	Tyr	Phe	Val	Arg	Tyr	Tyr	Tyr	Tyr	Tyr
		1	2	3	4	5	6	7	8	9	10	11	12	13
	D	AUG	UAU	UAU	UAU	UAU	UUC	GUG	CGU	UAU	UAU	UAU	UAU	UAU	F6(Cy3)-L11(Cy5)	0.73 ± 0.01 (Fig S2C)	0.43 ± 0.01 (Fig S2C)	0.69 ± 0.01 (Fig S2F)	0.87
		fMet	Tyr	Tyr	Tyr	Tyr	Phe	Val	Arg	Tyr	Tyr	Tyr	Tyr	Tyr
		1	2	3	4	5	6	7	8	9	10	11	12	13
mRNA-2,3	E	AUG	UUC	GUG	CGU	UAU	UAU	UAU	UAU	UAU	UAU	UAU	UAU	UAU	V3(Cy3)-L11(Cy5)	0.73 ± 0.01 (Fig S1J)	0.46 ± 0.02 (Fig S1J)	0.66 ± 0.01 (Fig S1M)	0.74
		fMet	Phe	Val	Arg	Tyr	Tyr	Tyr	Tyr	Tyr	Tyr	Tyr	Tyr	Tyr
		1	2	3	4	5	6	7	8	9	10	11	12	13
	F	AUG	UUC	GUG	CGU	UAU	UAU	UAU	UAU	UAU	UAU	UAU	UAU	UAU	V3(Cy5)-L11(Cy3)	0.70 ± 0.01 (Fig S1Q)	0.49 ± 0.03 (Fig S1Q)	0.63 ± 0.02 (Fig S1T)	0.67
		fMet	Phe	Val	Arg	Tyr	Tyr	Tyr	Tyr	Tyr	Tyr	Tyr	Tyr	Tyr
		1	2	3	4	5	6	7	8	9	10	11	12	13
	G	AUG	UUC	GUG	CGU	UAU	UAU	UAU	UAU	UAU	UAU	UAU	UAU	UAU	F2(Cy3)-L11(Cy5)	0.74 ± 0.01 (Fig S2L)	0.43 ± 0.01 (Fig S2L)	0.68 ± 0.01 (Fig S2O)	0.81
		fMet	Phe	Val	Arg	Tyr	Tyr	Tyr	Tyr	Tyr	Tyr	Tyr	Tyr	Tyr
		1	2	3	4	5	6	7	8	9	10	11	12	13

mRNA-6,7 and mRNA-2,3 displayed in the table were used to program unlabeled ribosomes (B and C), L11(Cy5) ribosomes (A, D, E, and G), and L11(Cy3) ribosomes (F) for smFRET experiments using the FRET pairs listed in the table, corresponding to the italicized (Cy5) or underlined (Cy3) codons in the sequences. PRE complexes were stalled by the absence of EF-G and used to form two-peaked FRET distributions fit with a double Gaussian function, giving the high and low FRET peaks corresponding to the classical and hybrid tRNA positions. The peak centers from aggregated data, as well as SEs of the peaks from multiple replicate experiments, are displayed in the table. Experiments were also conducted during ongoing translation in the presence of 2 μM EF-G, which yielded single-peaked FRET distributions of aggregate data, whose values are displayed in the table along with SEs from multiple replicate experiments. The single peaks (corresponding to INT_ongoing) had FRET efficiencies in between the stalled classical and hybrid values, as expressed in the ratio (E_ongoing – E_hybrid)/(E_classical – E_hybrid).