The location of mRNA in the ribosomal 30S initiation complex; site-directed cross-linking of mRNA analogues carrying several photo-reactive labels simultaneously on either side of the AUG start codon.
Messenger RNA molecules 30-35 bases long, with sequences related to the 5'-region of cro-mRNA from lambda-phage, were prepared by T7 transcription from synthetic DNA templates. Each mRNA contained five or six internal uridine residues, which were transcribed using a mixture of UTP and thio-UTP. Initiation complexes were formed with Escherichia coli 30S ribosomes in the presence or absence of tRNA(fMet), and cross-linking of the thio-U residues was induced by UV irradiation at wavelengths greater than 300 nm. The cross-linked ribosomal proteins were identified immunologically, and cross-linked regions of the 16S RNA were isolated by excision with ribonuclease H and suitable deoxyoligonucleotides. In both cases, the particular thio-U residue involved in the cross-link was identified by ribonuclease T1 fingerprinting of the (radioactive) mRNA in the isolated cross-linked complex. The principal results were that, at thio-U positions upstream of the AUG codon, specific cross-linking occurred to protein S7 and to the 3'-terminus of the 16S RNA, in agreement with similar experiments using 70S ribosomes. Less specific cross-linking was observed to proteins S1, S18 and S21 at various positions within the mRNA. Six bases downstream from the AUG codon, a tRNA-dependent cross-link was found to position approximately 1050 of the 16S RNA, but--in contrast to similar experiments with 70S ribosomes--no cross-linking was found to the 1390-1400 region.
Dontsova
O
O
Chemistry Department, Moscow State University, USSR.
Kopylov
A
A
Brimacombe
R
R
D001483:Base Sequence; D003062:Codon; D004247:DNA / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D004722:Endoribonucleases / Q000737:chemistry; D008969:Molecular Sequence Data; D010442:Peptide Chain Initiation, Translational; D010777:Photochemistry; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism*; D012336:RNA, Ribosomal, 16S / Q000378:metabolism; D016914:Ribonuclease H; D012269:Ribosomal Proteins / Q000378:metabolism*; D013698:Templates, Genetic; D014158:Transcription, Genetic
D003062:Codon; D012333:RNA, Messenger; D012336:RNA, Ribosomal, 16S; D012269:Ribosomal Proteins; C040469:ribosomal protein S30; D004247:DNA; D004722:Endoribonucleases; D016914:Ribonuclease H