escherichia coli ribosomal subunits , initiation factors , elongation factors were prepared and purified as described . trna and trna were labeled with fluorescent cyanine dyes at their elbow positions ( u8 or u47 ) , purified , and aminoacylated as previously described . a single - cysteine mutant of initiation factor 2 ( c599a and k791c ) was labeled with monomaleimide - cy5 ( ge lifesciences ) according to instructions from ge lifesciences . ribosome initiation complexes were assembled at 0.25 m 30s subunit concentration in a polymix buffer ( 50 mm tris - acetate ( ph 7.5 ) , 100 mm potassium chloride , 5 mm ammonium acetate , 0.5 mm calcium acetate , 5 mm magnesium acetate , 0.5 mm edta , 5 mm putrescine - hcl and 1 mm spermidine ) as described by uemura et al .. nucleotide concentration is at 4 mm for gtp , gdp , and gdpnp in all experiments . biotinylated messenger rnas were used to immobilize 30s pics with or without fmet - trna . complexes were tethered to the biotin - peg - derivatized quartz surface on the bottom of zmw wells through tetrameric neutravidin adaptor molecule by establishing peg - biotin : neutravidin : biotinylated - mrna complexes . the mrna used contains the following in order from 5 to 3 : a 5-utr and shine - dalgarno sequence derived from gene 32 of the t4 phage , an aug start codon , 6 repeats of phe - lys codons , an uaa stop codon , and 4 spacer phe codons ( supplementary figure 1 ) . immobilized pics were identified by initiator trna or 30s - subunit fluorescence and were distributed in zmw holes according to poisson statistics . a single photobleaching step for the scoring control experiments without mrna demonstrated the absence of non - specific surface interactions at concentrations up to 1m of labeled trnas , factors , or ribosomes . thus , fluorescent events observed here represent true interactions of translation components with immobilized 30s subunits . we delivered a mixture of fmet-(cy3)trna , cy5-if2(gtp ) , and cy3.5 - 50s to immobilized alexa488 - 30s at 20 nm , 200 nm , or 1000 nm of each reagent . the appearance of a stable 50s signal was used to identify productive trna and if2 binding events . the relative timing of if2 and trna arrival to the ribosome was determined by the order that their respective signals appear in each trace . observations for all experiments were done at 30 frames per second with ~33.3 ms exposure . therefore , simultaneous arrival of fluorescence signals can be either genuine simultaneous arrival events or the two events happening sequentially within the exposure time . from the trna and if2 arrival rates observed in control experiments , we calculated the percentage of apparent simultaneous events that can be attributed to sequential events happening in quick succession within 1 frame of exposure . we subtracted that part from the simultaneous events we observed and represented the subtracted portion as instrument uncertainty ,  or the gray portion on the bar graph in figure 1c . we delivered 200 nm of cy5 - 50s to immobilized cy3 - 30s at different magnesium concentataions ( 2.5~10 mm ) both with and without 1 m if2 in either gtp or gdpnp . where present , if1 and if3 are at 1m . the wait time until the appearance of the 50s signal and its lifetime was analyzed to determine the efficiency of subunit joining under the different conditions . we delivered 20~1000 nm cy5-if2 , 200~1000 nm cy3.5 - 50s and 200~1000 nm phe-(cy2)trna in a ternary complex with ef - tu and gtp to 30s pic loaded with fmet-(cy3)trna , simultaneously tracking 4 different labeled components in either gtp or gdpnp . we determined the overlap times of the if2 signal with the 50s signal and the relative timing of if2 departure to the arrival of the elongator trna , as well as the frequency of trna binding on the 70s ribosome post subunit joining . escherichia coli ribosomal subunits , initiation factors , elongation factors were prepared and purified as described . trna and trna were labeled with fluorescent cyanine dyes at their elbow positions ( u8 or u47 ) , purified , and aminoacylated as previously described . a single - cysteine mutant of initiation factor 2 ( c599a and k791c ) was labeled with monomaleimide - cy5 ( ge lifesciences ) according to instructions from ge lifesciences . ribosome initiation complexes were assembled at 0.25 m 30s subunit concentration in a polymix buffer ( 50 mm tris - acetate ( ph 7.5 ) , 100 mm potassium chloride , 5 mm ammonium acetate , 0.5 mm calcium acetate , 5 mm magnesium acetate , 0.5 mm edta , 5 mm putrescine - hcl and 1 mm spermidine ) as described by uemura et al .. nucleotide concentration is at 4 mm for gtp , gdp , and gdpnp in all experiments . biotinylated messenger rnas were used to immobilize 30s pics with or without fmet - trna . complexes were tethered to the biotin - peg - derivatized quartz surface on the bottom of zmw wells through tetrameric neutravidin adaptor molecule by establishing peg - biotin : neutravidin : biotinylated - mrna complexes . the mrna used contains the following in order from 5 to 3 : a 5-utr and shine - dalgarno sequence derived from gene 32 of the t4 phage , an aug start codon , 6 repeats of phe - lys codons , an uaa stop codon , and 4 spacer phe codons ( supplementary figure 1 ) . immobilized pics were identified by initiator trna or 30s - subunit fluorescence and were distributed in zmw holes according to poisson statistics . a single photobleaching step for the scoring control experiments without mrna demonstrated the absence of non - specific surface interactions at concentrations up to 1m of labeled trnas , factors , or ribosomes . thus , fluorescent events observed here represent true interactions of translation components with immobilized 30s subunits . we delivered a mixture of fmet-(cy3)trna , cy5-if2(gtp ) , and cy3.5 - 50s to immobilized alexa488 - 30s at 20 nm , 200 nm , or 1000 nm of each reagent . the appearance of a stable 50s signal was used to identify productive trna and if2 binding events . the relative timing of if2 and trna arrival to the ribosome was determined by the order that their respective signals appear in each trace . observations for all experiments were done at 30 frames per second with ~33.3 ms exposure . therefore , simultaneous arrival of fluorescence signals can be either genuine simultaneous arrival events or the two events happening sequentially within the exposure time . from the trna and if2 arrival rates observed in control experiments , we calculated the percentage of apparent simultaneous events that can be attributed to sequential events happening in quick succession within 1 frame of exposure . we subtracted that part from the simultaneous events we observed and represented the subtracted portion as instrument uncertainty ,  or the gray portion on the bar graph in figure 1c . we delivered 200 nm of cy5 - 50s to immobilized cy3 - 30s at different magnesium concentataions ( 2.5~10 mm ) both with and without 1 m if2 in either gtp or gdpnp . where present , if1 and if3 are at 1m . the wait time until the appearance of the 50s signal and its lifetime was analyzed to determine the efficiency of subunit joining under the different conditions . we delivered 20~1000 nm cy5-if2 , 200~1000 nm cy3.5 - 50s and 200~1000 nm phe-(cy2)trna in a ternary complex with ef - tu and gtp to 30s pic loaded with fmet-(cy3)trna , simultaneously tracking 4 different labeled components in either gtp or gdpnp . we determined the overlap times of the if2 signal with the 50s signal and the relative timing of if2 departure to the arrival of the elongator trna , as well as the frequency of trna binding on the 70s ribosome post subunit joining .
initiation of translation establishes the reading frame for protein synthesis and is a key point of regulation1 . 
 initiation involves factor - driven assembly at a start codon of an mrna of an elongation competent 70s ribosomal particle ( in bacteria ) from separated 30s and 50s subunits and initiator trna . 
 here we establish by direct single - molecule tracking the timing of initiator trna , initiation factor 2 ( if2 ) , and 50s subunit joining during initiation . 
 our results show multiple pathways to initiation , with orders of arrival of trna and if2 dependent on factor concentration and composition . 
 if2 accelerates 50s subunit joining , and stabilizes the assembled 70s complex . 
 transition to elongation is gated by the departure of if2 after gtp hydrolysis , allowing efficient arrival of elongator trnas to the second codon presented in the aminoacyl - trna acceptor site . 
 these experiments highlight the power of single - molecule approaches to delineate mechanism in complex multicomponent systems .