Homogeneous stalled ribosome nascent chain complexes produced in vivo or in vitro.
Cotranslational protein maturation is often studied in cell-free translation mixtures, using stalled ribosome-nascent chain complexes produced by translating truncated mRNA. This approach has two limitations: (i) it can be technically challenging, and (ii) it only works in vitro, where the concentrations of cellular components differ from concentrations in vivo. We have developed a method to produce stalled ribosomes bearing nascent chains of a specified length by using a 'stall sequence', derived from the Escherichia coli SecM protein, which interacts with residues in the ribosomal exit tunnel to stall SecM translation. When the stall sequence is expressed at the end of nascent chains, stable translation-arrested ribosome complexes accumulate in intact cells or cell-free extracts. SecM-directed stalling is efficient, with negligible effects on viability. This method is straightforward and suitable for producing stalled ribosome complexes in vivo, permitting study of the length-dependent maturation of nascent chains in the cellular milieu.
Evans
Michael S
MS
Department of Chemistry and Biochemistry, University of Notre Dame, 251 Nieuwland Science Hall, Notre Dame, Indiana 46556, USA.
Ugrinov
Krastyu G
KG
Frese
Marc-André
MA
Clark
Patricia L
PL
D001483:Base Sequence; D004926:Escherichia coli / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000235:genetics*; D014176:Protein Biosynthesis / Q000235:genetics*; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism*; D012270:Ribosomes / Q000378:metabolism*; D014157:Transcription Factors / Q000235:genetics*
D029968:Escherichia coli Proteins; D012333:RNA, Messenger; C497347:SecM protein, E coli; D014157:Transcription Factors