Tracing the path of messenger RNA on the Escherichia coli small ribosomal subunit. Immune electron microscopy using defined oligodeoxynucleotide analogs of mRNA.

Abstract

Oligodeoxynucleotide models of mRNA were used to determine the ribosomal site of specific nucleotides 3' to the initiation codon. Each mRNA analog had a 5'-terminal 9-base Shine-Dalgarno sequence, a 7-nucleotide spacer, and an ATG initiation signal, followed by up to 31 nucleotides, one of which carried an antibody-recognizable marker. All probes bound efficiently to activated Escherichia coli 30 S ribosomal subunits. Complexes were formed using ribosomal subunits, initiator tRNA, an oligodeoxynucleotide probe, and antibodies. Electron microscopy was then used to place specific positions in the mRNA analog on the subunit and thus to trace the pathway of the messenger. As viewed from the cytoplasmic side of the subunit, the 5' segment of the mRNA lies on the left, along the inner surface of the platform. The initiation codon and the next 9 nucleotides are located in the cleft between the subunit platform and body; within this segment the mRNA makes a U turn and emerges from the cleft at the left of the neck that separates the subunit head and body. The mRNA then loops around the neck to the right, along the cytoplasmic surface of the subunit and toward the site of the 7-methylguanosine residue of the 16 S ribosomal RNA.