Identification of the Escherichia coli 30S ribosomal subunit protein neighboring mRNA during initiation of translation

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 f Mei), 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 λ 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 condon in the P site. According to the second approach, 5-azidouridine (5N 3U) was randomly incorporated into mRNA transcripts during a T7 RNA polymerase catalyzed reaction by using a mixture of 5N 3UTP 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 5N 3U transcripts were essentially ‘zero-length’, the 5′-derivatized transcripts were covalently attached to ribosomal components about 14 Å from the 5'-end. We found the proteins S1, S7, S5, S3 and S4 compose, or were close to, the ribosomal mRNA-binding site. The 5′-end of the start codon might be within 14 Å from protein S7, while the 5′-end of the Shine-Dalgarno sequence probably has no definite binding site. Finally we discuss a possible way of mRNA positioning on the 30S ribosomal subunit.