N-Acetylvalyl-tRNAYl (AcVal-tRNA 1) was bound to the P site of uniformly 32P-labeled 70S ribosomes from Escherichia coli and crosslinked to 16S RNA in the 30S ribosomal subunit by irradiation with light of300-400 nm. To identify the crosslinked nucleotide in 16S RNA, AcVal-tRNAY. l-16S [32P]RNA was digested completely with RNase TI and the band containing the covalently attached oligonucleotides from tRNA and rRNA was isolated by polyacrylamide gel electrophoresis. The crosslinked oligonucleotide, and the 32P-labeled rRNA moiety released from it by photoreversal of the crosslink at 254 nm, were then analyzed by secondary hydrolysis with pancreatic RNase A and RNase U2. The oligonucleotide derived from 16S RNA was found to be the evolutionarily conserved sequence, U-A-C-A-C-A-C-C-G1401, and the nucleotide crosslinked to tRNAI , C14W. The identity of the covalently attached residue in the tRNA was established by using AcVal-tRNAlv-16S RNA prepared from unlabeled ribosomes.This complex was digested to completion with RNase T1 and the resulting RNA fragments were labeled at the 3' end with [5'-32P]pCp. The crosslinked Ti oligonucleotide isolated from the mixture yielded one major end-labeled component upon photoreversal. Chemical sequence analysis demonstrated that this product was derived from the anticodon-containing pentadecanucleo-
Ribosomal protein LI from the prokaryote Eacherichia coli has been shown to form a specific complex with 26S ribosomal RNA from the eukaryote Dictyostelium discoideum. The segment of Dictyostelium rRNA protected from ribonuclease digestion by LI and the corresponding region in Dictyostelium rDNA were investigated by nucleotide sequence analysis, and an analogous section in rDNA from Xenopus laevis was identified. When the LI-specific segments from eukaryotic rRNA were compared with those from prokaryotic rRNA, striking similarities in both primary and secondary structure were apparent. These conserved features suggest a common structural basis for protein recognition and indicate that such regions became fixed at a very early stage in rRNA evolution. In addition, certain structural elements ofthe LI binding sites in rRNA are also found in the initial segment of the polycistronic LI I-LI mRNA, providing support for the hypothesis that LI participates in the regulation of ribosomal protein synthesis by specific interaction with its own mRNA.Ribosomal particles are maintained in a compact, functionally active state by an intricate network of interactions among their protein and RNA constituents. Many ribosomal proteins exhibit high affinity for specific binding sites in rRNA, and stable complexes between purified components can be readily formed and studied in vitro (1). Protein LI from the 50S ribosomal subunit of Escherichia coli, for example, associates strongly and independently with homologous 23S RNA at unit stoichiometry (2) and protects a sequence of approximately 115 bases from ribonuclease digestion at its presumed attachment site (3). E. coli Li also has been found to form specific heterologous complexes with 23S RNAs from two species of Bacillus and four different archaebacteria (4, 5), which indicates that this interaction has been subject to strong evolutionary constraints within the prokaryotic domain. Although little is known about the function of Li in the ribosome, recent investigations suggest that it may be capable of binding not only to rRNA but also to the mRNA from which it is translated, thus serving as a "feedback repressor" that regulates the synthesis of proteins in the Lll-Li operon (6, 7).We report here the formation of a specific complex between protein Li from a prokaryote, E. coli, and 26S RNA from the large ribosomal subunit of a eukaryote, the slime mold Dictyostelium discoideum. Sequence analyses of Ll-associated fragments from Dictyostelium 26S RNA and the corresponding region ofDictyostelium rDNA and data on Li-specific segments from E. coli and Bacillus stearothermophilus 23S RNAs (3, 4) have allowed us to identify elements of primary and secondary structure common to all known Li binding sites. In addition, we have found that a similar region occurs in the 28S rRNA of a vertebrate, Xenopus laevis. Therefore, the structural features required for interaction with protein Li have been conserved in large-subunit rRNAs of both prokaryotes and eukaryotes despite the wide di...
Precursor tRNAAsp molecules, containing a 26-base 5' leader, were treated with diethylpyrocarbonate, 50% hydrazine or anhydrous hydrazine/3M NaCl and then subjected to processing by RNase P RNAs from Escherichia coli or Bacillus subtilis. Fully processed tRNAs and material not successfully cleaved by the catalytic RNAs were analyzed for their content of chemically altered nucleotides. Several bases were identified as being required intact for optimal activity as substrate as judged by exclusion of chemically modified residues from processed molecules, and simultaneous enhancement in material that was not recognized as substrate. Such nucleotides cluster near the site of cleavage at the mature 5' end and in the T stem and loop. Purines at residues 1 and 2 adjacent to the site of cleavage, position 57 in the T loop, and site 64 in the T stem exhibited the most pronounced effects. These results suggest a model of recognition of substrate by RNase P RNAs in which the ribozyme interacts with the corner of the precursor tRNA's three dimensional structure, where the T- and D-loops are juxtaposed, and extends along the top of the molecule back towards the site of catalysis.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.