ABSTRACT
Molecular modeling has been performed to help correct and refine an approximate three dimensional structure for Eschericia coli 16S rRNA that originated as hand-built wire model. The wire model was built to accommodate the base pairs in 16S rRNA (1), mRNA.16S rRNA interactions (2,3), rRNA.ribosomal protein interactions (see 4), ribosomal protein-ribosomal protein distances (5), intramolecular RNA-RNA UV-crosslinks (6,7) and site-directed mutagenesis experiments (8). In the computer model, individual base-paired regions were constructed separately and then were installed into the expected locations to produce the entire structure. We describe here the considerations, steps and results of this refinement process.
Subject(s)
Models, Molecular , RNA, Bacterial/chemistry , RNA, Ribosomal, 16S/chemistry , Computer Simulation , Escherichia coli/chemistry , Escherichia coli/genetics , Molecular Structure , Mutagenesis, Site-Directed , Nucleic Acid Conformation , RNA, Bacterial/genetics , RNA, Ribosomal, 16S/genetics , Ribosomes/chemistryABSTRACT
The arrangement of the mRNA on the Escherichia coli ribosome with respect to ribosomal RNA sites has been investigated by photochemical cross-linking experiments. mRNA analogues 51-54 nucleotides in length contained a Shine-Dalgarno sequence, a single codon for tRNA(Gly), and 4-thiouridine (s4U) in the 5' third of the mRNA (-20 to -12), in the middle third of the mRNA (-3 to +6), or in the 3' third of the mRNA (+20 to +26), where the position numbers are counted from the first nucleotide of the codon. Complexes were formed with these mRNAs and 70S ribosomes in the absence or presence of tRNA(Gly) and were irradiated. The extent of cross-linking and the identity of cross-linked rRNA sites were determined on agarose gels and by primer extension. 16S rRNA nucleotides A412, A532, G693 (weakly), U723, and U1381 (weakly) cross-linked with s4U in the 3' third; A532, G693, U723, A1167 (weakly), U1381, G818 (weakly), and A845 cross-linked with s4U in the middle; A532, G693, U723, A1167, G818 (weakly), and A845 cross-linked with s4U in the 5' third. All of these cross-links occur with tRNA independence. Cross-links at C1395 and A1196 occur for all three mRNAs with tRNA dependence. The pattern of these sites provides information about the order of the rRNA sites along the mRNA track, and they also point out the apparent overlapping neighborhoods for the mRNA track. Models for the track of the mRNA on the 30S subunit are considered to explain this pattern of interactions.
Subject(s)
Escherichia coli/metabolism , RNA, Messenger/metabolism , RNA, Ribosomal, 16S/metabolism , Ribosomes/metabolism , Base Sequence , Binding Sites , Codon , Cross-Linking Reagents , Electrophoresis, Agar Gel , Molecular Sequence Data , RNA, Messenger/chemistry , RNA, Messenger/isolation & purification , RNA, Ribosomal, 16S/chemistry , RNA, Ribosomal, 16S/isolation & purification , RNA, Transfer, Gly/biosynthesis , ThiouridineABSTRACT
Interactions between mRNA and rRNA on the 30S ribosomal subunit or 70S ribosome have been determined by photochemical cross-linking experiments using synthetic mRNA analogs substituted with 4-thiouridine. A set of RNA molecules containing different sequences has been used to determine the extent to which binding contacts are sequence dependent. The 16S rRNA and 23S rRNA nucleotides that form a part of the binding site have been identified by reverse transcription. The nucleotides are U1381, G1338, G1300, G1156, A845, U723, G693, A532, G497, U420, G413/A412, and G436 of 16S rRNA and U887 of 23S rRNA. Several additional nucleotides (U1065 of 23S rRNA and A1227, G818, G524, and G423 of 16S rRNA) are seen for some, but not all, of the mRNAs. Results obtained with two mRNAs containing the Shine-Dalgarno sequence were similar to those obtained with mRNAs lacking the Shine-Dalgarno sequence. Eight of these cross-linking sites were also seen when a mixture of RNA was used in which there are 12 random nucleotides preceding and seven random nucleotides succeeding an AUG codon. These results indicate that to a large extent placement of the mRNA in the ribosome does not depend upon its primary sequence.
