Initiation complexes formed by E. coli ribosomes in the presence of 32P-labeled A protein initiator region from R17 bacteriophage RNA have been treated with colicin E3 and disassembled by exposure to 1% sodium dodecyl sulfate. Electrophoresis on 9% polyacrylamide gels reveals a dissociabte complex containing the 30-nucleotide-long messenger fragment and the 50-nucleotide-long colicin fragment, which arises from the 3' terminus of the 16S RNA. The complex is a pure RNA-RNA hybrid; it is apparently maintained by a seven-base complementarity between the two RNA fragments.Detection of this mRNA rRNA complex strongly supports the hypothesis that during the initiation step of protein biosynthesis the 3' end of 16S RNA base pairs with the polypurine stretch common to initiator regions in E. coli and bacteriophage mRNAs. The implications of our findings with respect to the molecular mechanism of initiation site selection and mRNA binding to ribosomes, the role of rRNA in ribosome function, and species specificity in translation are explored.Shine and Dalgarno (1) originally suggested that a sequence near the 3' terminus of Escherschla coli 16S ribosomal RNA participates directly in the initiation of protein biosynthesis by forming several Watson-Crick base pairs with the messenger RNA. Indeed, one of the few common features of all ribosome-protected initiator regions analyzed so far is a polypurine stretch of 3 to 8 nucleotides located about 10 bases 5' to the initiator codon (Table 1). From 3 to 7 contiguous bases within this region of each mRNA can potentially pair with some portion of the polypyrimidine sequence found in the 3'-terminal Ti oligonucleotide of 16S RNA. Although the relevant 16S RNA sequence as determined by Shine and Dalgarno (1) conflicted with previous reports (2), its validity has now been confirmed in three additional laboratories (3-5).

We demonstrate the use of Lee-Goldburg cross-polarization (LG-CP) NMR under fast magic-angle spinning (MAS) to investigate the amplitude and geometry of segmental motions in biomolecular and polymeric solids. Motional geometry information was previously available only from 2 H NMR, which, however, has limited site resolution and requires site-specific isotopic labeling. Using a 2D LG-CP technique, we resolve the 13 C-1 H or 15 N-1 H dipolar couplings according to the 13 C or 15 N isotropic chemical shift. Applications to systems undergoing 180°phenylene ring flips show spectral line shapes reflecting the geometry of the motion. Using this LG-CP technique, we measured the 13 C-1 H and 15 N-1 H dipolar couplings in the water-soluble and membrane-bound states of the colicin Ia channel domain. The backbone motions of the membrane-bound colicin scale both the CR-HR and N-H couplings similarly, thus ruling out rotation of the R-helices around their axes as a specific mechanism of motion. We also show that the sensitivity of the LG-CP spectra can be enhanced by the addition of a phase-inverted 1 H-13 C cross-polarization step, and the site resolution of the 15 N-1 H LG-CP spectra can be enhanced by 13 C indirect detection.

The selective and extensive 13C labeling of mostly hydrophobic amino acid residues in a 25 kDa membrane protein, the colicin Ia channel domain, is reported. The novel 13C labeling approach takes advantage of the amino acid biosynthetic pathways in bacteria and suppresses the synthesis of the amino acid products of the citric acid cycle. The selectivity and extensiveness of labeling significantly simplify the solid-state NMR spectra, reduce line broadening, and should permit the simultaneous measurement of multiple structural constraints. We show the assignment of most 13C resonances to specific amino acid types based on the characteristic chemical shifts, the 13C labeling pattern, and the amino acid composition of the protein. The assignment is partly confirmed by a 2D homonuclear double-quantum-filter experiment under magic-angle spinning. The high sensitivity and spectral resolution attained with this 13C-labeling protocol, which is termed TEASE for ten-amino acid selective and extensive labeling, are demonstrated.

Voltage-gated channels undergo a conformational change in response to changes in transmembrane voltage. Here we use site-directed biotinylation to create conformation-sensitive sites on colicin Ia, a bacteriocidal protein that forms a voltage-sensitive membrane channel, which can be monitored by electrophysiological methods. We investigated a model of gating developed for the partly homologous colicin E1 that is based on the insertion of regions of the protein into the membrane in response to cis-positive voltages. Site-directed cysteine mutagenesis, followed by chemical modification, was used to attach a biotin molecule covalently to a series of unique sites on colicin Ia. The modified protein was incorporated into planar lipid membranes, where the introduced biotin moiety served as a site to bind the water-soluble protein streptavidin, added to one side of the membrane or the other. Our results show that colicin gating is associated with the translocation across the membrane of a segment of the protein of at least 31 amino acids.