e. coli strain bearing hairpin loop extensions in phylogenetically - variable , surface - accessible loops of 16s rrna in helix 44 and 23s rrna in helix 101 were used to purify 70s ribosomes .
3-dye labeled dna oligonucleotides ( labeled with cy3b or bhq-2 ) complementary to the mutant ribosome hairpins can then be used to label the ribosomes .
translation factors ( if2 , ef - tu , ef - g , ef - ts ) , s1 , and fluorescently labeled cy5-ef - g , were prepared as described previously for our single - molecule experiments .
a semi - purified mix of all aminoacyl - trna synthetases ( aars ) was prepared from e. coli s30 extract .
total or (lys , val , phe , or some combination ) aminoacyl - trnas were prepared by incubating total e. coli trna ( sigma - aldrich ) with all amino acids ( minus lys , val , phe or some combination for  mixes ) together with the aars mix for 30 min at 37c in a buffer consisting of tris - hcl ( 50 mm , ph 7.5 ) , kcl ( 50 mm ) , mgcl2 ( 10 mm ) , and -me ( 3 mm ) , complemented with atp ( 2 mm ) , phosphoenol pyruvate ( pep , 10 mm ) , pyruvate kinase ( pk , 50 g / ml ) , myokinase ( mk , 2 g / ml ) and inorganic pyrophosphatase ( ppiase , 10 g / ml ) .
aa - trnas were thereafter purified by phenol extraction , ethanol precipitation , and gel filtration ( micro bio - spin columns with bio - gel p-6 in tris buffer , bio - rad ) , and finally stored in small aliquots in 80c .
phe-(cy5)trna and lys-(cy5)trna were labeled with cy5-nhs ( ge lifesciences ) at acpu at position 47 , purified as previously described , and aminoacylated as above .
val-(cy3)trna was labeled with cy3-maleimide ( ge lifesciences ) at su at position 8 , purified and aminoacylated as above .
all mrnas had 5-biotin followed by a 5'-utr and shine - dalgarno sequence derived from gene 32 of the t4 phage ( sequence described previously ) , an aug start codon , followed by the sequence of interest ( as indicated in the figures ) .
all experiments were conducted in a tris - based polymix buffer consisting of 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 .
3'-dye labeled dna oligonucleotides ( labeled with cy3b or bhq-2 ) complementary to the mutant ribosome hairpins were ordered from trilink .
right before each experiment , purified 30s and 50s ribosomal subunits ( final concentration = 1 m ) were mixed in 1:1 ratio with the 3 dye - labeled oligonucleotides specific for the hairpin extensions in each subunit for 37c for 10 min and then at 30c for 20 min in a tris - based polymix buffer system .
30s pre - initiation complexes ( pics ) were formed as described by incubating the following at 37 c for 5 minutes : 0.25 m cy3b-30s , pre - incubated with stoichiometric s1 , 1 m if2 , 1 m fmet - trna , 1 m mrna , and 4 mm gtp to form 30s pics in the polymix buffer . before use ,
we pre - incubate a smrt cell v3 from pacific biosciences ( menlo park , ca , usa ) , a zero - mode waveguide ( zmw ) chip , with a 1 mg / ml neutravidin solution in 50 mm tris - acetate ph 7.5 and 50 mm kcl at room temperature for 5 minutes . the cell is then washed with buffer 6 ( 50 mm tris - acetate ph 7.5 , 100 mm potassium chloride , 5 mm ammonium acetate , 0.5 mm calcium acetate , 5 mm magnesium acetate , and 0.5 mm edta ) .
after washing , 40 l of buffer 6 is left in the cell to keep the cell surface wet .
we then dilute the 30s pics with our tris - based polymix buffer containing 1 m if2 and 4 mm gtp down to 10 nm pic concentration .
the diluted pics are then loaded into the smrt cell at room temperature for 3 minutes to immobilize the 30s pics into the zmw wells .
we wash away excessive unbound material with our tris - based polymix buffer containing 1 m if2 , 4 mm gtp , 1 mm trolox , and a pca / pcd oxygen scavenging system ( 2.5 mm 3,4-dihydroxybenzoic acid and 250 nm protocatechuate deoxygenase ) . after washing ,
20 l of the fresh washing mix were added to the cell to keep the surface wet and to prevent exposure of the immobilized dye labeled biomolecules to oxygen .
we formed ternary complexes ( tcs ) between total charged e. coli trnas and ef - tu(gtp ) as described .
total or (aa ) aminoacyl - trnaef - tugtp ternary complexes were pre - formed by incubating ( 2 min at 37c ) the aa - trnas with five - fold excess of ef - tu , gtp ( 1 mm ) , pep ( 3 mm ) and ef - ts ( 40 m ) in polymix . the ternary complexes ( 1 ~ 6 m )
were added to bhq-50s ( 200 nm ) , ef - g ( 80 ~ 480 nm ) , if2 ( 1 m ) , gtp ( 4 mm ) , 2.5 mm trolox , and the oxygen scavenging system ( pca / pcd ) to form a delivery mix in polymix buffer .
many of the experiments are done at 1 m ternary complexes and 80 nm ef - g ( chosen to have well - defined , detectable fret transition signals ) , unless indicated otherwise . before starting an experiment ,
the smrt cell is loaded into a modified pacbio rs sequencer . at the start of the elongation experiment
, the instrument illuminates the smrt cell with a green laser and then delivers 20 l of a delivery mixture onto the cell surface at t ~ 10 sec .
translation experiments using labeled trnas are performed in the same way as ribosome cy3b / bhq conformational fret experiments with the following differences .
total trna mixture was charged with all amino acids except the trna amino acid we wish to observe . the resulting mix of all charged trnas except trna of interest that remains uncharged was used to form ternary complexes .
for example , we will form a ternary complex with phe-(cy5)trna and (phe ) aa - trna .
the delivery mix contains : 200 nm bhq-50s , 1 m if2 , 80~320 nm ef - g , 1~3 m total trna aa ternary complexes , 200 nm aa-(cy3/cy5)trna ternary complexes , 4 mm gtp , 1 mm trolox , and the pca / pcd oxygen scavenging system in the tris - based polymix buffer .
all experiments are performed at 200 nm labeled - trna ternary complexes . during the experiment ,
the experiments involving trna transit of cy3-trna ( 200 nm ) and cy5-trna ( 200 nm ) are done the same way , except the ribosomes were not labeled and a vallys aa - trna ( 3 m ) mixture is used with 80 ~ 320 nm ef - g .
cross - correlation experiments between our ribosome cy3b / bhq conformational fret signal and our ef - g binding signal are conducted exactly as above but with a different delivery mix and laser illumination .
the delivery mix for the cross - correlation experiment contains : 200 nm bhq250s , 1 m if2 , 80 ~ 240 nm cy5-ef - g , 1 ~ 3 m total trna ternary complexes , 4 mm gtp , 1 mm trolox , and the pca / pcd oxygen scavenging system in tris - based polymix buffer .
the data presented in figure 4 are performed at 80 nm cy5-ef - g and 1 m total trna ternary complexes .
all single - molecule fluorescence experiments were conducted using a commercial pacbio rs sequencer that we modified to allow the collection of single - molecule fluorescence intensities from individual zmw wells about 130 nm in diameter in 4 different dye channels corresponding to cy3 , cy3.5 , cy5 , and cy5.5 .
the rs sequencer has two lasers for dye excitation at 532 nm and 632 nm . in all experiments ,
data was collected at 10 frames per second ( 100 ms exposure time ) for 8 minutes .
the energy flux of the green laser is 0.48 w/m and the red laser is at 0.22 w/m .
data analyses for all experiments are conducted with matlab ( mathworks ) scripts written in - house .
briefly , traces from the zmw wells are initially selected based on fluorescence intensity , fluorescence lifetime , and the changes in intensity .
filtered traces exhibiting intersubunit fret or single - molecule binding signals are then selected for further data analysis .
the fret states are assigned as previously described based on a hidden markov model based approach and visually corrected .
all lifetimes were fitted to a single - exponential distribution using maximum - likelihood parameter estimation in matlab .
e. coli strain bearing hairpin loop extensions in phylogenetically - variable , surface - accessible loops of 16s rrna in helix 44 and 23s rrna in helix 101 were used to purify 70s ribosomes .
3-dye labeled dna oligonucleotides ( labeled with cy3b or bhq-2 ) complementary to the mutant ribosome hairpins can then be used to label the ribosomes .
translation factors ( if2 , ef - tu , ef - g , ef - ts ) , s1 , and fluorescently labeled cy5-ef - g , were prepared as described previously for our single - molecule experiments .
a semi - purified mix of all aminoacyl - trna synthetases ( aars ) was prepared from e. coli s30 extract .
total or (lys , val , phe , or some combination ) aminoacyl - trnas were prepared by incubating total e. coli trna ( sigma - aldrich ) with all amino acids ( minus lys , val , phe or some combination for  mixes ) together with the aars mix for 30 min at 37c in a buffer consisting of tris - hcl ( 50 mm , ph 7.5 ) , kcl ( 50 mm ) , mgcl2 ( 10 mm ) , and -me ( 3 mm ) , complemented with atp ( 2 mm ) , phosphoenol pyruvate ( pep , 10 mm ) , pyruvate kinase ( pk , 50 g / ml ) , myokinase ( mk , 2 g / ml ) and inorganic pyrophosphatase ( ppiase , 10 g / ml ) .
aa - trnas were thereafter purified by phenol extraction , ethanol precipitation , and gel filtration ( micro bio - spin columns with bio - gel p-6 in tris buffer , bio - rad ) , and finally stored in small aliquots in 80c .
phe-(cy5)trna and lys-(cy5)trna were labeled with cy5-nhs ( ge lifesciences ) at acpu at position 47 , purified as previously described , and aminoacylated as above .
val-(cy3)trna was labeled with cy3-maleimide ( ge lifesciences ) at su at position 8 , purified and aminoacylated as above .
all mrnas had 5-biotin followed by a 5'-utr and shine - dalgarno sequence derived from gene 32 of the t4 phage ( sequence described previously ) , an aug start codon , followed by the sequence of interest ( as indicated in the figures ) .
all experiments were conducted in a tris - based polymix buffer consisting of 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 .
3'-dye labeled dna oligonucleotides ( labeled with cy3b or bhq-2 ) complementary to the mutant ribosome hairpins were ordered from trilink .
right before each experiment , purified 30s and 50s ribosomal subunits ( final concentration = 1 m ) were mixed in 1:1 ratio with the 3 dye - labeled oligonucleotides specific for the hairpin extensions in each subunit for 37c for 10 min and then at 30c for 20 min in a tris - based polymix buffer system .
30s pre - initiation complexes ( pics ) were formed as described by incubating the following at 37 c for 5 minutes : 0.25 m cy3b-30s , pre - incubated with stoichiometric s1 , 1 m if2 , 1 m fmet - trna , 1 m mrna , and 4 mm gtp to form 30s pics in the polymix buffer . before use ,
we pre - incubate a smrt cell v3 from pacific biosciences ( menlo park , ca , usa ) , a zero - mode waveguide ( zmw ) chip , with a 1 mg / ml neutravidin solution in 50 mm tris - acetate ph 7.5 and 50 mm kcl at room temperature for 5 minutes . the cell is then washed with buffer 6 ( 50 mm tris - acetate ph 7.5 , 100 mm potassium chloride , 5 mm ammonium acetate , 0.5 mm calcium acetate , 5 mm magnesium acetate , and 0.5 mm edta ) .
after washing , 40 l of buffer 6 is left in the cell to keep the cell surface wet .
we then dilute the 30s pics with our tris - based polymix buffer containing 1 m if2 and 4 mm gtp down to 10 nm pic concentration .
the diluted pics are then loaded into the smrt cell at room temperature for 3 minutes to immobilize the 30s pics into the zmw wells .
we wash away excessive unbound material with our tris - based polymix buffer containing 1 m if2 , 4 mm gtp , 1 mm trolox , and a pca / pcd oxygen scavenging system ( 2.5 mm 3,4-dihydroxybenzoic acid and 250 nm protocatechuate deoxygenase ) . after washing ,
20 l of the fresh washing mix were added to the cell to keep the surface wet and to prevent exposure of the immobilized dye labeled biomolecules to oxygen .
we formed ternary complexes ( tcs ) between total charged e. coli trnas and ef - tu(gtp ) as described .
total or (aa ) aminoacyl - trnaef - tugtp ternary complexes were pre - formed by incubating ( 2 min at 37c ) the aa - trnas with five - fold excess of ef - tu , gtp ( 1 mm ) , pep ( 3 mm ) and ef - ts ( 40 m ) in polymix . the ternary complexes ( 1 ~ 6 m )
were added to bhq-50s ( 200 nm ) , ef - g ( 80 ~ 480 nm ) , if2 ( 1 m ) , gtp ( 4 mm ) , 2.5 mm trolox , and the oxygen scavenging system ( pca / pcd ) to form a delivery mix in polymix buffer .
many of the experiments are done at 1 m ternary complexes and 80 nm ef - g ( chosen to have well - defined , detectable fret transition signals ) , unless indicated otherwise . before starting an experiment ,
the smrt cell is loaded into a modified pacbio rs sequencer . at the start of the elongation experiment
, the instrument illuminates the smrt cell with a green laser and then delivers 20 l of a delivery mixture onto the cell surface at t ~ 10 sec .
translation experiments using labeled trnas are performed in the same way as ribosome cy3b / bhq conformational fret experiments with the following differences .
total trna mixture was charged with all amino acids except the trna amino acid we wish to observe . the resulting mix of all charged trnas except trna of interest that remains uncharged was used to form ternary complexes .
for example , we will form a ternary complex with phe-(cy5)trna and (phe ) aa - trna .
the delivery mix contains : 200 nm bhq-50s , 1 m if2 , 80~320 nm ef - g , 1~3 m total trna aa ternary complexes , 200 nm aa-(cy3/cy5)trna ternary complexes , 4 mm gtp , 1 mm trolox , and the pca / pcd oxygen scavenging system in the tris - based polymix buffer .
all experiments are performed at 200 nm labeled - trna ternary complexes . during the experiment ,
the smrt cell is illuminated with both a green and a red laser . the experiments involving trna transit of cy3-trna ( 200 nm ) and cy5-trna ( 200 nm )
are done the same way , except the ribosomes were not labeled and a vallys aa - trna ( 3 m ) mixture is used with 80 ~ 320 nm ef - g .
cross - correlation experiments between our ribosome cy3b / bhq conformational fret signal and our ef - g binding signal are conducted exactly as above but with a different delivery mix and laser illumination .
the delivery mix for the cross - correlation experiment contains : 200 nm bhq250s , 1 m if2 , 80 ~ 240 nm cy5-ef - g , 1 ~ 3 m total trna ternary complexes , 4 mm gtp , 1 mm trolox , and the pca / pcd oxygen scavenging system in tris - based polymix buffer .
the data presented in figure 4 are performed at 80 nm cy5-ef - g and 1 m total trna ternary complexes .
all single - molecule fluorescence experiments were conducted using a commercial pacbio rs sequencer that we modified to allow the collection of single - molecule fluorescence intensities from individual zmw wells about 130 nm in diameter in 4 different dye channels corresponding to cy3 , cy3.5 , cy5 , and cy5.5 .
the rs sequencer has two lasers for dye excitation at 532 nm and 632 nm . in all experiments ,
data was collected at 10 frames per second ( 100 ms exposure time ) for 8 minutes .
the energy flux of the green laser is 0.48 w/m and the red laser is at 0.22 w/m .
data analyses for all experiments are conducted with matlab ( mathworks ) scripts written in - house .
briefly , traces from the zmw wells are initially selected based on fluorescence intensity , fluorescence lifetime , and the changes in intensity . filtered traces exhibiting intersubunit fret or single - molecule binding signals
the fret states are assigned as previously described based on a hidden markov model based approach and visually corrected .
all lifetimes were fitted to a single - exponential distribution using maximum - likelihood parameter estimation in matlab .
frameshifting have been proposed to occur at each of the steps : ( 1 ) during accommodation of the a - site trna , ( 2 ) subsequent to accommodation , but prior to peptide bond formation , ( 3 ) during trna hybrid - state intermediates , ( 4 ) during translocation , and ( 5 ) at the start of the next found of elongation .
( b ) the ribosome starts each round of elongation in the nonrotated state . in this  locked  state , the p - site trna is stably bound in the classical state , preserving the reading frame of the mrna . upon a - site
trna selection and peptide bond formation , the 30s subunit rotates 3~10 counterclockwise with respect to the 50s subunit to the rotated state ( pre - translocation ) .
state permits trna motions and the trna can fluctuate freely between the classical state and hybrid state , thus facilitating translocation of trna and movement of ribosome by one codon over mrna .
peptide - bond formation also triggers spontaneous fluctuations of the l1 stalk between open and closed conformations as well as spontaneous rotations in ribosome conformations .
ef - g then catalyzes translocation , and the ribosome returns to the nonrotated state ( post - translocation ) . to monitor the rotational state of the ribosome in real time , we employed fret between the small ( 30s ) and large ( 50s ) subunits .
the 30s subunit was site - specifically labeled with cy3b on helix 44 , and a nonfluorescent quencher , bhq-2 , was placed on helix 101 of 50s subunit .
reagent delivery of bhq-50s , trna ternary complex , and ef - g to surface immobilized cy3b-30s pre - initiation complexes in zmws results in if2-guided 70s assembly during initiation and establishment of fret between the two ribosomal subunits : upon subunit joining , the green ( cy3b ) intensity drops , which is followed by alternating low - high - low intensities each alternating cycle corresponds to the ribosome translating a single codon , with the two intensity states consistent with the two rotational states of the ribosome : the low intensity state ( high fret ) defining the nonrotated ( locked ) ribosome conformation and the high intensity state ( low fret ) the rotated ( unlocked ) conformation .
( c ) during each cycle of elongation , the ribosome selects the aminoacyl - trna in a ternary complex with ef - tu - gtp , and positions the trna in the a site . upon a - site trna accommodation ,
translocation moves a- and p - site trna - mrna complexes to the e and p site respectively , catalyzed by the ef - g .
the compositional dynamics of trna and ef - g on the ribosome , here defined as the relative timing of their arrival and departure during elongation , can be observed by labeling the trna or ef - g with cy3 or cy5 .
cy5/cy3-trna arrival to the surface immobilized ribosomes is marked by a red / green fluorescent pulse .
each productive trna binding event results in a fluorescence pulse that lasts as a ribosome translates 2 codons  beginning with arrival of trna in the a - site , continuing through trna translocation to the p - site , arrival of a - site trna to the next codon , a second round of translocation , and ending with spontaneous dissociation of trna from the e site . to track trna and ef - g dynamics on translating ribosomes at near - physiological concentrations of fluorescent factors ( 0.1  1 m ) , we used zmws to detect hundreds of individual ribosomes ( d ) substituting the traditional fret acceptor , cy5 , with bhq-2 allowed the use of cy5 to label other translation components for correlation studies .
the cy3b intensity reports on the conformational state of the ribosome , while cy5 pulses indicate arrival , occupancy , and departure of ribosomal ligands .
( a ) histogram of the fraction of ribosomes translating to a particular codon for the dnax 1 frameshift mrna , with a schematic .
many of the ribosomes translate up to 12 codons where the 0 frame stop codon is , though a large percentage of ribosomes translate up to 9 codons , where the 1 frame stop codon is .
there are also ribosomes that stall at codon 7 , limited by cy3b photobleaching or end of movie ( 8 minutes ) from the long rotated state pause .
interestingly , there are also a significant number of ribosomes that stall at codon 8 ( see extended data fig . 10 for discussion ) . by parsing the number of ribosomes that translate beyond codon 9 and up to codon 9 ,
the frameshifting percentage can be calculated ( 75% ) . however , since non - frameshifted ribosomes may terminate early , this would lead to a slight over - estimate of the frameshifting percentage ( 3~10% ) .
the frameshifting efficiency has been independently confirmed using a cy5-trna score , as described below .
( b ) a uuc(phe ) codon is introduced in the 1 frame downstream of the slippery site .
frameshifting can be scored by an appearance of a cy5 ( red ) pulse with cy5-trna in addition to the cy3b / bhq conformational fret signal .
( c ) using the cy5-trna as a score to confirm frameshifting , we get the same dynamics and lifetimes : the nonrotated state lifetimes remain constant at each codon , and the rotated state lifetime increases 10-fold at codon lys7 at the slippery sequence .
( d ) by using the cy5-trna as a score , we can parse the rotated state lifetimes into ribosomes that frameshifted and ribosomes that did not frameshift .
we also get the same results as figure 1 : non - frameshifted ribosomes translate through the frameshift sequence seemingly unaffected ; frameshifted ribosomes exhibit the characteristic long - rotated state pause at codon lys7 .
the mrna consists of the same sequence as the wild - type dnax frameshift sequence , but with the sequence after the uga stop codon in the 0 frame in the hairpin deleted .
( b ) nonrotated and rotated state lifetimes in the presence of 80 nm ef - g and 1 m trnatot .
( c ) mrna sequence of the no shine - dalgarno ( no sd ) mutant .
the mrna consists of the same sequence as the wild - type dnax frameshift sequence , but with the original internal shine - dalgarno sequence gggagc mutated to aggcgc , decreasing the rrna - mrna interaction energy from 3.00 kj / mol to 0.06 kj / mol .
( d ) nonrotated and rotated state lifetimes in the presence of 80 nm ef - g and 1 m trnatot .
( e ) frameshifting percentages of the no sd and no hp mutant . without the shine - dalgarno sequence , frameshifting percentage drops by half . without the hairpin , frameshifting percentage drops to a quarter of the wild - type sequence .
this indicates that both the internal shine - dalgarno sequence and the hairpin are required for efficient frameshifting , confirming that the hairpin and internal sd are stimulatory elements for frameshifting .
these stimulatory elements may present a barrier and tension to translocation that is a prerequisite for efficient frameshifting .
( a ) translation of a short linear mrna , 6(fk ) , in the presence of 80 nm ef - g and 1 m trnatot , with an example trace .
ribosomes translate < 12 codons are due to photobleaching of the cy3b dye , or non - processive ribosomes .
the small number of ribosomes that translate beyond codon 12 are likely errors in our statistical analysis or read - through of the stop codon .
( d ) translation of a phe - lys sequence preceded by an internal shine - dalgarno sequence ( same sd sequence used in the dnax frameshift mrna of this study ) in the presence of 80 nm ef - g and 1 m trnatot , with an example trace .
( f ) there is an increase in rotated state lifetime at codon 5 ~ 7 .
there is an increase in the rotated state lifetimes 3~4 fold over 3~5 codons downstream of the sd - like sequence , while the non - rotated state lifetime remains unaffected .
the internal sd - like sequences may base pair with the 3 end of the 16s rrna and slow down ribosomes in the pre - translocation state , echoing several work done previously by tracking ribosome movement and ribosome profiling .
( g ) translation of a phe - lys sequence followed by a hairpin ( same hairpin used in the dnax frameshift mrna of this study ) in the presence of 80 nm ef - g and 1 m trnatot , with an example trace .
there is an increase in rotated state lifetime at codon 5 , exactly 3 codons before the start of the hairpin , placing the ribosome directly at the first encounter of the hairpin .
the relative position also matches where we see the long - rotated state pause during frameshift .
this echoes the work done by tinoco et al . where they showed the ribosome is capable of translating through the secondary structure through two mechanisms : ribosome translocating when encountering an open - state junction , occurring naturally or induced by the ribosome , or mechanically unwinding by the ribosome when encountering a closed - state junction .
when the ribosome encounters an open - state junction , translation proceeds at a constant rate ; however , when a closed - state junction is encountered , the ribosome actively unwinds the secondary structure , resulting in a slight waiting time for translocation , after which the hairpin is biased by the ribosome into an open - state and translation occurs normally .
the shunt to either pausing in the rotated state ( which leads to uncoupled translocation ) or normal translation during frameshifting is likely due to this mechanism .
( a ) example trace and schematic of a ribosome translating the dnax frameshift mrna at much higher factor concentrations ( 6 m trnatot and 480 nm ef - g ) .
( b ) frameshifting efficiency does not depend on ef - g and trnatot concentrations .
( c ) increasing the trnatot and ef - g concentrations two - fold ( from 1 m trnatot and 80 nm ef - g to 2 m trnatot and 160 nm ef - g ) decreases both the rotated state lifetime and nonrotated state lifetimes .
the lifetime of the long rotated state pause at codon lys7 is also decreased by half , indicating that ef - g and/or trna interacts with the ribosome in that state .
( d ) increasing the trnatot concentration ( from 1 m to 3 m ) while keeping ef - g concentrations constant decrease the non - rotated state lifetimes 3-fold as expected .
the rotated state lifetimes remain the same except for codon lys7 ; this is expected because the rotated state lifetime depends only on concentration of ef - g .
unexpectedly , the rotated state lifetime at codon lys7 is also slightly decreased , suggesting a linkage between trna and ef - g dynamics at that long rotated - state stall .
this echoes our results in figure 2 that trna ( trna in this case ) samples the uncoupled rotated - state after translocation , and the trna sampling and accommodation may help to re - establish the ribosome s reading frame and reverse - rotate subsequently .
thus , increasing trna concentrations ( especially trna in this case ) will decrease the long rotated - state lifetime .
( e ) increasing the ef - g concentration ( from 80 nm to 240 nm ) while keeping trnatot concentration constant decreases the rotated state lifetime 3-fold .
the nonrotated state lifetime , which depends on the trna concentration , remains the same .
however , the decrease in the rotated lifetime at codon lys7 is only around 2-fold , rather than 3-fold as expected .
this echoes our result in figure 2 as well as part ( b ) above , suggesting that trna sampling also plays a role at this codon .
( f ) increasing the ef - g and trnatot concentrations further to 6 m trnatot and 480 nm ef - g further decreases the rotated and nonrotated state lifetimes . however , the rotated state lifetime at codon lys7 remains the same when compared with 2 m trnatot and 160 nm ef - g .
the long trna sampling events observed in figure 2 may be contributing to the long rotated state lifetime at codon lys7 .
( a ) example trace of a ribosome translating the a21ga24 g mutant mrna in the presence of 80 nm ef - g and 1 m trnatot
( b ) histogram of the fraction of ribosomes translating to a particular codon for the dnax 1 frameshift a21ga24 g mrna .
most of the ribosomes translate up to 12 codons where the 0 frame stop codon is . the buildup of ribosomes stalled at codon 9 present during frameshifting disappears . by parsing the number of ribosomes that translate beyond codon 9 and up to codon 9 ,
the long stall at lys7 is decreased with the slippery site mutant , suggesting that the extra - long pause is indeed a result of frameshifting .
the slight increase in lifetime at lys7 is due to the effects of the hairpin and internal shine - dalgarno sequence .
( d ) a uuc(phe ) is introduced in the 1 frame downstream of the slippery site of the a21ga24 g mutant , similar to above .
( e ) the nonrotated state lifetime and rotated - state lifetime match with our results using codon counting ( see above ) . in the absence of frameshifting
, there is still an increase in rotated state lifetime at codon lys7 , due to the increased energy barrier to translocation by the hairpin and internal shine - dalgarno sequence , though this increased lifetime is still much less than the lys7 rotated state lifetime during frameshifting .
( f ) using cy5-trna as a score for frameshifting , frameshifting percentage matches with our previous results . the slippery sequence a21ga24 g mutant decreases frameshifting percentage down to background levels .
( a ) the 3 nucleotides upstream of the slippery sequence ( gca(ala ) ) is mutated to gua(val ) ( named the c20u mutant ) so that e - site trna dynamics can be observed during frameshifting since trna can be labeled with cy3-maleimide ( see figure 2 ) .
this allows us to estimate the time to translocation during the long rotated - state pause at codon lys7 , since translocation of the cy3-trna from the p - site to the e - site leads to rapid departure of the trna and disappearance of the cy3 signal .
the nonrotated state and rotated state lifetimes , as well as frameshifting percentages , are consistent with what we have observed before for the wild - type sequence .
( b ) trna - trna fret between the cy3-trna in the p site and the incoming cy5-trna at lys7 in the a site at the slippery sequence .
the trnas are in a hybrid state upon encountering of hairpin and engagement with the internal shine - dalgarno sequence .
after translocation , the cy3-trna departs from the ribosome , resulting in a disappearance of fret .
after translocation and uncoupling with ribosome reverse - rotation , the now p - site trna is likely in a hybrid , or  distorted 
( a ) example trace of cy5-trna transit during translation of the dnax frameshift mrna , indicating the definition of pulse lifetime and time between pulse .
( b ) the time between trna pulse and lifetime of each pulse for the first three lys codons are consistent with what is expected , and decrease expectedly with the increase of ef - g concentration and trnatotal(lys ) concentration ( [ cy5-trna]= 200 nm ) .
the time between pulse for the first lys pulse corresponds to the decoding of the first codon lys1 of the mrna after 50s subunit joining to the immobilized 30s , which is short as expected and does not depend on factor concentration .
the second lys pulse has a longer time between pulse , corresponding to the ribosome translating four codons from lys1 to lys5 .
the third pulse has a slightly shorter time between pulse , corresponding to the ribosome translating from codon lys5 to lys7 .
the lifetimes for the first two pulses are short , since the ribosomes decode and translocate the corresponding codons normally .
the lifetime for the third pulse at codon lys7 is long , corresponding to the ribosome at the long rotated - state pause during frameshifting .
( c ) mean number of additional trna sampling pulses to the long rotated - state pause at codon lys7 , sampling lifetimes , and sampling arrival times , at various concentrations of ef - g and trnatot ( [ cy5-trna ] = 200 nm ) for ribosomes translating the dnax 1 wild - type frameshifting sequence .
there is a mean number of ~2.3 sampling trna pulses , which remains constant at the various factor concentrations .
there seems to be an interplay ( competition ) between ef - g and the other trnas on trna sampling . increasing the concentration of other factors increase the arrival time for trna , probably because they are all competing for the ribosomal a site .
number of molecules analyzed , from left to right , n = 179 , n = 212 , n = 180 , n = 162 .
( d ) the time between trna pulse and lifetime of each pulse for the first three lys codons are the same for the wild - type aag sequence and the aag(aaa ) mutant sequence .
( e ) by translating the aag(aaa ) mutant in the presence of cy5-trna , we see similar dynamics as the dnax wild - type sequence .
cy5-trna samples the a site at codon lys8 after uncoupled translocation at the frameshift site .
the fraction of ribosomes exhibiting > 4 trna pulses are the same for the wild - type sequence and the aag(aaa ) mutant .
the mean number of trna sampling pulses , the mean arrival time , and the mean lifetimes of the sampling pulses to the long rotated - state stall are the same for the aag(aaa ) mutant and the dnax wild - type sequence .
( a ) example traces of cy5-trna ( red ) sampling to the a - site phe8 codon during the long rotated - state pause correlated with cy3b / bhq conformational fret signal ( green ) for the aag(uuu ) mutant .
( b ) by translating the aag(uuu ) mutant in the presence of cy5-trna ( red ) and correlating with the cy3b / bhq conformational fret signal ( green ) , we can observe the fraction of ribosomes exhibiting only 1 cy5-trna pulse or > 1 cy5-trna pulse sampling to the long rotated state pause at codon lys7 .
there is a significant number of ribosomes exhibiting > 1 cy5-trna pulse even when there is only one phe codon , suggesting that even without frameshifting , many of the ribosomes still pause in an uncoupled rotated state at lys7 , where trna samples the exposed uuu codon in the a site .
( c ) the arrival time of the first trna sampling to the long stalled codon for wild - type mrna ( with cy5-trna ) and aag(uuu ) with cy5-trna are the same .
although frameshifting in principle could occur through an incomplete + 2 translocation with weakened codon - anticodon - ribosome interactions and the final reading - frame determined through the lys8 trna sampling in the 1 frame , our data support + 3 translocation that weakens codon - anticodon - ribosome interactions followed by trna sampling and accommodation that defines the reading frame and shifts 1 through effects of the hairpin and internal - sd interaction . for + 2 translocation , we would expect to see trna sampling to both 1 frame and the 0 frame of a - site codon .
for the aag(uuu ) mutant , trna will sample the 0 frame u25u26u27 , while trna will sample the 1 frame a24u25u26 . since , cy5-trna arrival times and lifetimes for sampling to the aag(uuu ) mutant match cy5-trna arrival times and lifetimes for the wild - type sequence , there is likely no competition between trna and trna suggesting that the auu codon is not initially exposed for trna sampling .
furthermore , for the + 2 model , we would not expect a aag(uuu ) mutation to lead to a decrease in frameshifting efficiency .
thus , our results favor a + 3 translocation followed by a 1 shift driven by sampling , accommodation and base - pairing stability .
unfortunately , our single - molecule assay is blind to the actual movement of the ribosome on the mrna , so the details of this mechanism will require further exploration .
( d ) mean sampling lifetime and mean sampling arrival time for cy5-trna to the phe8 codon for the aag(uuu ) mutant .
the arrival time and lifetime are the same as cy5-trna sampling to the lys8 codon for the dnax wild - type sequence .
( e ) example trace for cy5-trna transit through the dnax frameshift mrna aag(uuu ) mutant . for the aag(uuu )
mutant , we see only three lys pulses , as expected , since the fourth lys codon ( lys8 ) is mutated to a phe codon .
most of the ribosomes ( ~80% ) exhibit only three cy5-trna pulses , indicating that the additional cy5-trna sampling pulses we saw characteristic of frameshifting are indeed sampling to the lys8 codon .
the time between pulses are consistent with both the wild - type sequence and aag(aaa ) mutant .
the lifetime of the third pulse ( at lys7 ) is long , consistent with the long pause at that codon .
( f ) example trace for cy5-trna transit through the dnax frameshift mrna aag(aac ) mutant . for the aag(aac )
mutant , we see mostly only three lys pulses ( ~75% ) since the fourth lys codon ( lys8 ) is mutated to a asn codon .
this further argues against the + 2 translocation model ( see part ( c ) ) . for + 2 translocation , we would expect to see long lys - trna sampling to the 1 frame aaa codon , which is not observed significantly .
the additional cy5-trna pulses have a shorter lifetime and longer arrival time when compared with the translation of the wild - type mrna , suggesting that these pulses are non - cognate sampling to the aac codon in the 0 frame or sampling unstably to the aaa codon in the 1 frame .
even though our data supports a + 3 translocation followed by a 1 slippage , multiple frameshifting pathways probably occurs .
( a ) two different protein products are possible after 1 frameshifting , dependent on whether peptide bond formation occurs during sampling in the 1 or 0 frame .
for the first scenario , trna sampling to the last three nucleotides of the slippery sequence ( yyz ) redefines the ribosome in the 1 frame ( yyy ) , after which the trna dissociates to leave an empty a - site codon .
after the long rotated state is reverse - rotated by ef - g , trna decodes that codon normally , creating a frameshift product denoted by xxy - yyy . for the second scenario ,
peptide bond formation occurs after slippage of trna into the 1 frame ; peptide - bond formation occurs slowly , since the p- and a - site trnas would likely not be positioned correctly in the rotated ribosomal conformation .
ef - g would then normally and rapidly resolve the newly - created a / p hybrid state and the ribosome reverse - rotates . in this case
( b ) histogram of the fraction of ribosomes translating to a particular codon for the dnax 1 frameshift aag(uuu ) mrna , with a schematic .
since the frameshifting percentage for the aag(uuu ) sequence is low , we see that most of the ribosomes translate up to 12 codons where the 0 frame stop codon is .
though , there is a significant number of ribosomes that translate to 11 codons ( ~25% ) , compared to ~5% for our previous experiments .
there are two possible scenarios for trna sampling to the long rotated - state pause to codon phe8 . in the first case
, the trna defines the reading frame and falls off , after which the ribosome resolves itself through the action of ef - g , followed by the normal decoding of phe codon at codon 8 . in this case
, we get 12 cycles of low - high - low fret intensity , and hence 12 codons translated by our signal . in the second case
, the trna defines the reading frame , followed by slow peptide bond formation . after peptide bond formation , the ribosome returns to the canonical hybrid and rotated state , for which ef - g then catalyzes reverse rotation . in this case ,
one low - high - low fret cycle is missed , so we count 11 codons translated by our signal .
( c ) example traces of cy5-trna ( red ) sampling to the long rotated - state pause at codon lys7 correlated with cy3b / bhq conformational fret signal ( green ) , showing the two possible scenarios for trna sampling .
case 1 ( as described in part ( a ) ) leads to correlation of trna arrival and ribosome rotation after the long rotated state pause whereas case 2 leads to overlap of a trna pulse with the reverse - rotation of the long pause .
both scenarios occur when translating the aag(uuu ) mutant , with ~58% of ribosomes for case 1 and 42% for case 2 .
for the 2 case , the time between the last cy5-trna arrival and the ribosome reverse - rotation is 27.2 s , much longer than the 7.7 s during normal decoding and translocation , suggesting a slow peptidyltransfer reaction .
our results provide a possible explanation for why heterogeneous frameshifting products are observed in many frameshifting systems .
 spontaneous changes in the reading frame of translation are rare ( frequency of 103  104 per codon)1 , but can be induced by specific features in the messenger rna ( mrna ) . in the presence of mrna secondary structures , a heptanucleotide  slippery sequence  usually defined by the motif x xxy yyz , and ( in some prokaryotic cases ) mrna sequences that base pair with the 3 end of the 16s ribosomal rrna ( internal shine - dalgarno ( sd ) sequences ) , there is an increased probability that a specific programmed change of frame occurs , wherein the ribosome shifts one nucleotide backwards into an overlapping reading frame ( 1 frame ) and continues by translating a new sequence of amino acids2,3 . despite extensive biochemical and genetic studies , there is no clear mechanistic description for frameshifting . here 
 , we apply single - molecule fluorescence to track the compositional and conformational dynamics of the individual ribosomes at each codon during translation of a frameshift - inducing mrna from the dnax gene in escherichia coli . 
 ribosomes that frameshift into the 1 frame are characterized by a 10-fold longer pause in elongation compared to non - frameshifted ribosomes , which translate through unperturbed . during the pause , 
 interactions of the ribosome with the mrna stimulatory elements uncouple ef - g catalyzed translocation from normal ribosomal subunit reverse - rotation , leaving the ribosome in a non - canonical intersubunit rotated state with an exposed codon in the aminoacyl - trna site ( a site ) . 
 trnalys sampling and accommodation to the empty a site either lead to the slippage of the trnas into the 1 frame or maintain the ribosome into the 0 frame . 
 our results provide a general mechanistic and conformational framework for 1 frameshifting , highlighting multiple kinetic branchpoints during elongation . 