Following translation by single ribosomes one codon at a time.
We have followed individual ribosomes as they translate single messenger RNA hairpins tethered by the ends to optical tweezers. Here we reveal that translation occurs through successive translocation--and-pause cycles. The distribution of pause lengths, with a median of 2.8 s, indicates that at least two rate-determining processes control each pause. Each translocation step measures three bases--one codon-and occurs in less than 0.1 s. Analysis of the times required for translocation reveals, surprisingly, that there are three substeps in each step. Pause lengths, and thus the overall rate of translation, depend on the secondary structure of the mRNA; the applied force destabilizes secondary structure and decreases pause durations, but does not affect translocation times. Translocation and RNA unwinding are strictly coupled ribosomal functions.
Wen
Jin-Der
JD
Department of Chemistry, University of California, Berkeley, California 94720, USA.
Lancaster
Laura
L
Hodges
Courtney
C
Zeri
Ana-Carolina
AC
Yoshimura
Shige H
SH
Noller
Harry F
HF
Bustamante
Carlos
C
Tinoco
Ignacio
I
D049148:Aminoacylation; D020029:Base Pairing; D003062:Codon / Q000235:genetics*; D007700:Kinetics; D052898:Optical Tweezers; D014176:Protein Biosynthesis / Q000502:physiology*; D012333:RNA, Messenger / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012343:RNA, Transfer / Q000235:genetics / Q000378:metabolism; D012270:Ribosomes / Q000378:metabolism*; D013997:Time Factors
D003062:Codon; D012333:RNA, Messenger; D012343:RNA, Transfer