Identification of the Escherichia coli 30S ribosomal subunit protein neighboring mRNA during initiation of translation.
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)
Dontsova
O A
OA
Department of Chemistry, Moscow State University, Russia.
Rosen
K V
KV
Bogdanova
S L
SL
Skripkin
E A
EA
Kopylov
A M
AM
Bogdanov
A A
AA
D000345:Affinity Labels; D001386:Azides; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics; D001483:Base Sequence; D003432:Cross-Linking Reagents; D004926:Escherichia coli / Q000235:genetics*; D008969:Molecular Sequence Data; D010442:Peptide Chain Initiation, Translational / Q000235:genetics*; D012329:RNA, Bacterial / Q000737:chemistry* / Q000235:genetics; D012333:RNA, Messenger / Q000737:chemistry* / Q000235:genetics; D012269:Ribosomal Proteins / Q000737:chemistry* / Q000235:genetics; D013698:Templates, Genetic; D014544:Uridine Triphosphate / Q000031:analogs & derivatives
D000345:Affinity Labels; D001386:Azides; D001426:Bacterial Proteins; D003432:Cross-Linking Reagents; D012329:RNA, Bacterial; D012333:RNA, Messenger; D012269:Ribosomal Proteins; C040469:ribosomal protein S30; C072279:5-azidouridine-5'-triphosphate; D014544:Uridine Triphosphate