Structural analysis of the peptidyl transferase region in ribosomal RNA of the eukaryote Xenopus laevis.
Accessible single-strand bases in Xenopus laevis 28 S ribosomal RNA (rRNA) Domain V, the peptidyl transferase region, were determined by chemical modification with dimethylsulfate, 1-cyclohexyl-3-(2-morpholinoethyl-carbodiimide metho-p-toluene sulfonate and kethoxal, followed by primer extension. The relative accessibilities of three rRNA substrates were compared: deproteinized 28 S rRNA under non-denaturing conditions (free 28 S rRNA), 60 S subunits and 80 S ribosomes. Overall, our experimental results support the theoretical secondary structure model of Domain V derived by comparative sequence analysis and compensatory base-pair changes, and support some theoretical tertiary interactions previously suggested by covariation. The 60 S subunits and 80 S ribosomes generally show increasing resistance to chemical modification. Bases which are sensitive in free 28 S rRNA but protected in 60 S subunits may be sites for ribosomal protein binding or induced structural rearrangements. Another class of nucleotides is distinguished by its sensitivity in 60 S subunits but protection in 80 S ribosomes; these nucleotides may be involved in subunit-subunit interactions or located at the interface of the ribosome. We found a third class of bases, which is protected in free 28 S rRNA but sensitive in 60 S subunits and/or 80 S ribosomes, suggesting that structural changes occur in Domain V as a result of subunit assembly and ribosome formation. One such region is uniquely hypersensitive in eukaryotic ribosomes but is absent in Escherichia coli ribosomes. Sites that we determined to be accessible on empty 80 S ribosomes could serve as recognition sites for translation components.
Stebbins-Boaz
B
B
Division of Biology and Medicine, Brown University, Providence, RI 02912.
Gerbi
S A
SA
D000447:Aldehydes / Q000494:pharmacology; D000818:Animals; D001483:Base Sequence; D002074:Butanones; D003028:CME-Carbodiimide / Q000031:analogs & derivatives*; D002234:Carbodiimides / Q000494:pharmacology; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D010458:Peptidyl Transferases / Q000378:metabolism*; D012339:RNA, Ribosomal, 28S / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D012269:Ribosomal Proteins / Q000378:metabolism; D012270:Ribosomes / Q000378:metabolism; D013379:Substrate Specificity; D013463:Sulfuric Acid Esters / Q000494:pharmacology; D014982:Xenopus laevis
D000447:Aldehydes; D002074:Butanones; D002234:Carbodiimides; D012339:RNA, Ribosomal, 28S; D012269:Ribosomal Proteins; D013463:Sulfuric Acid Esters; C074111:N-cyclohexyl-N'-2-morpholinoethyl-carbodiimide-methyl-4-toluolsulfonate; D003028:CME-Carbodiimide; C005135:kethoxal; D010458:Peptidyl Transferases; C007482:dimethyl sulfate