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

O. Dontsova, A. Kopylov, R. Brimacombe

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91 Citations (Scopus)

Abstract

Messenger RNA molecules 30 - 35 bases long, with sequences related to the 5'-region of cro-mRNA from λ-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(f)(Met), and cross-linking of the thio-U residues was induced by UV irradiation at wavelengths >300 nm. The cross-linked ribosomal proteins were identified immunologically, and crosslinked 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 crosslinking 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 ~1050 of the 16S RNA, but-in contrast to similar experiments with 70S ribosomes-no cross-linking was found to the 1390-1400 region.

Original languageEnglish
Pages (from-to)2613-2620
Number of pages8
JournalEMBO Journal
Volume10
Issue number9
DOIs
Publication statusPublished - 1991
Externally publishedYes

Keywords

  • 30S initiation complex
  • mRNA analogues
  • Ribonuclease H digestion
  • Ribosomal protein antibodies Site-directed cross-linking

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