title: Ribosome initiation complex formation with the pseudoknotted alpha operon messenger RNA.
abstract: The Escherichia coli alpha 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 37 degrees C (5 mM-Mg2+, 100 mM-Na+, pH 7.0). A broad transition with tm approximately 38 degrees 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-tRNAf(met)-alpha 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 tRNAf(met), respectively; the active form is predominant above 37 degrees 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 alpha mRNA. The intact pseudoknot stimulates 30 S subunit binding, at low temperatures, but prevents proper binding of tRNAf(met). The inactive to active transition of the pseudoknot, which may be related to the 38 degrees C transition seen in melting experiments, is required for tRNAf(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.
authors.affiliation: Department of Chemistry, Johns Hopkins University, Baltimore, MD 21218.
authors.forename: G
authors.initials: G
authors.lastname: Spedding
authors.forename: T C
authors.initials: TC
authors.lastname: Gluick
authors.forename: D E
authors.initials: DE
authors.lastname: Draper
mesh_terms: D001483:Base Sequence; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D007700:Kinetics; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D009876:Operon; D014176:Protein Biosynthesis; D012329:RNA, Bacterial / Q000737:chemistry / Q000378:metabolism; D012333:RNA, Messenger / Q000737:chemistry* / Q000378:metabolism; D012358:RNA, Transfer, Met / Q000378:metabolism; D012270:Ribosomes / Q000378:metabolism*; D013816:Thermodynamics
chemical_list: D012329:RNA, Bacterial; D012333:RNA, Messenger; D012358:RNA, Transfer, Met