PMID:1379079

Citation	Dontsova, OA, Rosen, KV, Bogdanova, SL, Skripkin, EA, Kopylov, AM and Bogdanov, AA (1992) Identification of the Escherichia coli 30S ribosomal subunit protein neighboring mRNA during initiation of translation. Biochimie 74:363-71
Abstract	To identify the proteins of the 30S ribosomal subunit of E coli that neighbor mRNA in the ternary initiation complex (mRNA*30S subunit*tRNA(fMet), we used an affinity cross-linking approach in which photoactivated groups were attached to different positions along the mRNA chain. A series of mini-genes originating from the 5'-end region of the cro gene of lambda bacteriophage were constructed as templates for mini-mRNA synthesis. Two strategies were used to introduce photo-reactive agents into the message. According to the first, two transcripts were isolated from E coli and chemically derivatized at their 5'-ends with a photoinducible diaziril group. One of these messages allowed for localization of the 5'-end of the Shine-Dalgarno sequence while the other one allowed for labeling of the ribosome at the 5'-end side of the initiation AUG codon in the P site. According to the second approach, 5-azidouridine (5N3U) was randomly incorporated into mRNA transcripts during a T7 RNA polymerase catalyzed reaction by using a mixture of 5N3UTP and UTP. A message that had U residues at either -4, -3, -1, +2 and +14, +19, +20 positions was used (A from cro AUG is +1). Whereas cross-links with the 5N3U transcripts were essentially 'zero-length', the 5'-derivatized transcripts were covalently attached to ribosomal components about 14 A from the 5'-end. We found that proteins S1, S7, S5, S3 and S4 compose, or were close to, the ribosomal mRNA-binding site.(ABSTRACT TRUNCATED AT 250 WORDS)
Links	PubMed
Keywords	Affinity Labels; Azides; Bacterial Proteins/chemistry; Bacterial Proteins/genetics; Base Sequence; Cross-Linking Reagents; Escherichia coli/genetics; Molecular Sequence Data; Peptide Chain Initiation, Translational/genetics; RNA, Bacterial/chemistry; RNA, Bacterial/genetics; RNA, Messenger/chemistry; RNA, Messenger/genetics; Ribosomal Proteins/chemistry; Ribosomal Proteins/genetics; Templates, Genetic; Uridine Triphosphate/analogs & derivatives
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