Abstract
Photoinduced cross-linking of Escherichia coli 70 S ribosomes with [3H]puromycin has led to the labeling of ribosomal proteins S7, S14, S18, L18, and L29. Proteolytic fragmentation of these proteins and separation of the peptide mixtures by C18 reversed-phase high performance liquid chromatography resulted in six puromycin-labeled peptides which were applied to sequence analysis. The following peptides were found labeled: Pro1-Lys10 of S7, Ala28-Lys46 and Ala7-Lys11 of S14, Asp24-Lys29 of S18, Tyr64-Lys68 of L18, and Thr55-Lys60 of L29. For the first time the molecular environment of an antibiotic in the procaryotic ribosome is presented at the peptide level.
Highlights
Ribosomeswith [3H]puromycinhas led to the labeling of In this study we have identified peptide regions of small a n d ribosomal proteins S7, S14, SlS, LlS, and L29
Proteo- large ribosomal subunit proteins cross-linked to puromycin in lytic fragmentation ofthese proteins and separationof t h e Escherichia coli ribosome by direct sequence analysis.The the peptide mixtures by C, reversed-phase high per- results present forthe first time detailsof peptide regionsthat formance liquid chromatography resulted in six puro- are located at or near the peptidyltransferase center.The pepmycin-labeledpeptides which were appliedto sequence tides found cross-linked to puromycin are discussed with reanalysis
This reaction catalyzes the transfer [8-3HlPuromycin waps repared from puromycin(Sigma)by refluxing of the group peptidyl ester of peptidyl-tRNA of an incoming aminoacyl-tRNA
Summary
Ribosomeswith [3H]puromycinhas led to the labeling of In this study we have identified peptide regions of small a n d ribosomal proteins S7, S14, SlS, LlS, and L29. The final pellet was redissolved in TKMlO buffer (340 A,,, unitdml), Puromycin can be cross-linked ittos functional site and proteins were extracted for 5 min with 50% acetic acid (Nicholson by U V irradiation (Jaynes et al, 1978).
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
