EFG-independent translocation of the mRNA:tRNA complex is promoted by modification of the ribosome with thiol-specific reagents.
Translation of polyphenylalanine from a polyuridine template by the ribosome in the absence of the elongation factors EFG and EFTu (and the energy derived from GTP hydrolysis) is promoted by modification of the ribosome with thiol-specific reagents such as para-chloromercuribenzoate (pCMB). Here, we examine the translational cycle of modified ribosomes and show that peptide bond formation and tRNA binding are largely unaffected, whereas translocation of the mRNA:tRNA complex is substantially promoted by pCMB modification. The translocation movements that we observe are authentic by multiple criteria including the processivity of translation, accuracy of movement (three-nucleotide) along a defined mRNA template and sensitivity to antibiotics. Characterization of the modified ribosomes reveals that the protein content of the ribosomes is not depleted but that their subunit association properties are severely compromised. These data suggest that molecular targets (ribosomal proteins) in the interface region of the ribosome are critical barriers that influence the translocation of the mRNA:tRNA complex.
Southworth
Daniel R
DR
Department of Molecular Biology and Genetics, Johns Hopkins University School of Medicine, Howard Hughes Medical Institute, Baltimore, MD 21205, USA.
Brunelle
Julie L
JL
Green
Rachel
R
D000900:Anti-Bacterial Agents / Q000494:pharmacology; D001665:Binding Sites; D007700:Kinetics; D008274:Magnesium / Q000378:metabolism; D009843:Oligoribonucleotides / Q000378:metabolism; D020653:Peptide Elongation Factor G / Q000502:physiology; D010455:Peptides / Q000378:metabolism; D010458:Peptidyl Transferases / Q000378:metabolism; D011485:Protein Binding; D014176:Protein Biosynthesis; D021122:Protein Subunits / Q000187:drug effects / Q000378:metabolism; D011691:Puromycin / Q000494:pharmacology; D034443:RNA Transport; D012333:RNA, Messenger / Q000378:metabolism*; D012343:RNA, Transfer / Q000378:metabolism*; D012345:RNA, Transfer, Amino Acid-Specific / Q000378:metabolism; D012360:RNA, Transfer, Phe / Q000378:metabolism; D012269:Ribosomal Proteins / Q000378:metabolism; D012270:Ribosomes / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D013439:Sulfhydryl Reagents / Q000494:pharmacology*; D013997:Time Factors; D020245:p-Chloromercuribenzoic Acid / Q000494:pharmacology*
D000900:Anti-Bacterial Agents; D009843:Oligoribonucleotides; D020653:Peptide Elongation Factor G; D010455:Peptides; D021122:Protein Subunits; D012333:RNA, Messenger; D012345:RNA, Transfer, Amino Acid-Specific; D012360:RNA, Transfer, Phe; D012269:Ribosomal Proteins; D013439:Sulfhydryl Reagents; C012001:polyphenylalanine; D011691:Puromycin; D020245:p-Chloromercuribenzoic Acid; D012343:RNA, Transfer; D010458:Peptidyl Transferases; D008274:Magnesium