RIBOSOME INITIATION COMPLEX-FORMATION WITH THE PSEUDOKNOTTED ALPHA OPERON MESSENGER-RNA

The Escherichia coli α mRNA has a complex pseudoknot secondary structure that forms the recognition site for a translational repressor, ribosomal protein S4, and also encompasses the regulated ribosome binding site. To find out whether the pseudoknot is a stable structure under the conditions of ribosome initiation complex formation, thermal denaturation of the RNA was monitored by calorimetry and ultraviolet light hyperchromicity. The secondary structure formed by the coding region melts in a single transition and has a stability of -7.4 kcal/mol at 377#x00B0;C (5 mM-Mg2+, 100 mM-Na+, pH 7.0). A broad transition with t(m) ∼ 387#x00B0;C may be a rearrangement of pseudoknot secondary or tertiary structure. Using reverse transcriptase primer extension assays (’toeprints’) to measure the kinetics of ternary 30 S subunit-tRNA(f)(met)-α mRNA translational initiation complex formation, we find a fast and a slow phase in the reaction. The fraction reacting rapidly is sensitive to temperature and mutations in the mRNA. We interpret these results in terms of ’active’ and ’inactive’ mRNA conformations that are trapped by 30 S subunits and react rapidly or slowly with tRNA(f)(met), respectively; the active form is predominant above 377#x00B0;C. The binary 30 S-mRNA complex in the inactive form stops MMLV reverse transcriptase near the 3’ edge of the pseudoknot structure, apparently by stabilizing the pseudoknot. We propose the following mechanism for translational initiation with the α mRNA. The intact pseudoknot stimulates 30 S subunit binding, at low temperatures, but prevents proper binding of tRNA(f)(met). The inactive to active transition of the pseudoknot, which may be related to the 387#x00B0;C transition seen in melting experiments, is required for tRNA(f)(met) to pair with the anticodon and is rate-limiting for initiation complex formation at lower temperatures. A novel feature of this proposal is that the mRNA structure affects a kinetic step in initiation complex formation, as well as ribosome binding affinity. © 1993 Academic Press, Inc.