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The RNA helix destabilizing properties of Escherichia coli initiation factor 3 protein (IF3), and its affinity for an evolutionarily conserved sequence at the 3' end of 16S rRNA, led us to examine the details of the protein-nucleic acid interactions upon IF3 binding to the 49-nucleotide 3'-terminal cloacin DF13 fragment of 16S rRNA by studying the circular dichroism (CD) and proton magnetic resonance spectra of the RNA, the protein, and their complex. In a physiological tris(hydroxymethyl)aminomethane buffer, where the interaction is primarily nonionic and sequence specific, addition of IF3 decreases the RNA 268-nm CD peak hyperbolically by 19% to an end point of about one IF3 per RNA strand. The titration curve is best fit by an association constant of (1.80 +/- 0.05) X 10(7) M-1, within the range estimated by a nuclease mapping study of the same system [Wickstrom, E. (1983) Nucleic Acids Res. 11, 2035-2052]. In a low-salt phosphate buffer without Mg2+, where the interaction is primarily ionic and nonspecific, titration with IF3 decreases the peak CD sigmoidally by 35% to an end point of two IF3 per strand. The titration curve is best fit by an intrinsic association constant of (1.7 +/- 0.7) X 10(6) M-1 for each IF3 and a cooperativity constant of 33 +/- 6. In a physiological phosphate buffer lacking Mg2+, the dispersion of aromatic proton magnetic resonance peaks and upfield-shifted methyl proton resonances indicates a high degree of secondary and tertiary structure in the protein. In an equimolar mixture of IF3 and RNA cloacin fragment, several changes in identifiable IF3 and RNA resonances are observed.(ABSTRACT TRUNCATED AT 250 WORDS)

The effect of bacteriocin (cloacin DF13) treatment of Escherichia coli ribosomes on initiation of protein synthesis has been studied in detail. In agreement with our previous findings [Baan et al. (1976) Proc. Natl Acad. Sci. U.S.A. 73, 702--706] it is shown that 70-S initiation complexes can be formed with cloacin-treated ribosomes, but that the initiation factor IF-1 does not function properly. The following pleiotropic effects of this factor have been studied: (a) the acceleration of ribosomal subunit exchange with 70-S couples; (b) the stimulation of the IF-3-mediated dissociation of 70-S ribosomes; (c) the stimulation of 30-S initiation complex formation; (d) the enhancement of the rate of release of IF-2 from 70-S initiation complexes. The effects (a) and (b) are virtually abolished after cleavage of 16-S rRNA. The effect (d) is only partially reduced whereas effect (c) seems to be unimpaired. It is concluded that 70-S initiation complex formation with cloacin-treated ribosomes suffers from improper functioning of IF-1 in the generation of active subunits from 70-S tight couples. This is the only effect on initiation. It can be compensated for by adding more IF-3. The data provide functional evidence that 16-S rRNA is involved in ribosomal subunit interaction.

1 Mg2+-dependent dissociation of Escherichia coli ribosomes has been studied at various Mg2+ concentrations ranging from 10 to 0.5 mM. The extent of dissociation was determined by sucrose gradient centrifugation. 2 Of all 70 S ribosomes present in standard iS30 extracts about 50% dissociate in a relatively narrow range of Mg2+ concentration (between 4 and 2 mM), whereas the remainder dissociate between 10 and 4 mM Mg2+. 3 Puromycin at a concentration (0.1 mg/ml) which releases about 90% of nascent polypeptide chains after polypeptide synthesis does not influence the Mg2+-dependent dissociation curve of iS30 ribosomes, whereas it has a significant effect on the dissociation of 70 S ribosomes bearing peptidyl-tRNA (so called complexed 70 S ribosomes). Very high concentrations of puromycin (1 mg/ml) in addition to chain release also cause dissociation of ribosomes at 10 mM Mg2+. 4 Twenty-five per cent of the complexed 70 S particles, isolated after MS2-RNA programmed polypeptide synthesis, dissociate between 4 and 2 mM Mg2+. The remainder 75% dissociate between 2 and 0.5 mM Mg2+. 5 The distribution of the various classes of free and complexed 70 S ribosomes in iS30 extracts before and after MS2-RNA directed polypeptide synthesis and before and after puromycin action has been determined.

The effect of the presence or absence of methyl groups on the N6 atoms of two adjacent adenosines near the 3' end of 16 S rTNA of Escherichia coli on initiation of protein biosynthesis has been studied using wild type (methylated) and kasugamycin-resistant (unmethylated) E. coli ribosomes (see preceding paper (Poldermans, B., Goosen, N., and Van Knippenberg, P. H. (1979) J. Biol. Chem. 254, 9085--9089)). Conditions of pH, temperature, and ionic strength at which binding of fMet-tRNA to ribosomes proceeds maximally are the same for wild type and mutant ribosomes. Mg2+- and factor-dependent dissociation of ribosomes as well as the association of the subunits is also the same for methylated and unmethylated ribosomes. Binding of fMet-tRNA to wild type and to mutant 70 S ribosomes requires the same amount of the three initiation factors. However, optimal fMet-tRNA binding to unmethylated 30 S ribosomes needs more of initiation factor 3 than does binding to methylated 30 S ribosomes, provided that initiation factor 1 is absent. This difference is completely abolished when mutant 30 S ribosomes are methylated using purified methylase from the wild type strain and the methyl donor S-adenosylmethionine.

The "colicin" fragments comprising the 49 3'-terminal nucleotides of 16 S ribosomal RNA have been isolated from wild-type Escherichia coli and from a kasugamycin-resistant mutant that lacks methylation of two geminal adenine residues. Proton nuclear magnetic resonance (n.m.r.) spectra (500 MHz) were recorded at various temperatures. The low-field resonances arising from the hydrogen-bonded iminoprotons of paired bases were assigned using the nuclear Overhauser effect (n.o.e.). Crucial to the interpretation of the spectra are the resonances that originate from the two hydrogen-bonded iminoprotons of a U X G basepair. Combined with temperature-jump relaxation kinetics experiments the n.o.e.s lead to the conclusion that a conserved A X U/U X G junction in the hairpin is a thermolabile dislocation in the helix. The n.m.r. spectra of the wild-type and mutant fragment are only different with respect to the iminoproton resonances of the two base-pairs adjoining the hairpin loop. The spectra recorded at various temperatures tend to indicate that dimethylation of the adenosines labilizes these base-pairs, but no definitive conclusions are drawn. The results confirm our previous views that dimethylation of the adenosine residues affects the conformation of the hairpin loop.

Poly(uridylic acid) [poly(U)] and poly(xanthidylic acid) [poly(X)] strongly stimulate the IF-2-dependent binding of fMET-tRNA to 30-S ribosomal subunits from Escherichia coli [Van der Laken et al. (1979) FEBS Lett. 100, 230-234]. The N-formylmethionine moiety is incorporated into poly(phenylalanine) upon subsequent addition of other components required for protein synthesis when poly(U) is used as template. This paper shows that N-acetylated Phe-tRNAPhe (AcPhe-tRNA), but not Phe-tRNAPhe or tRNAPhe, competes with fMET-tRNA for binding to poly(U)-programmed 30-S ribosomal subunits. The two species of N-blocked aminoacyl-tRNA are bound to poly(U)-programmed and poly(X)-programmed 30-S subunits in a ratio that is linearly dependent on the ratio of the two species added. With poly(U) as template there is no apparent preference for either fMET-tRNA or AcPhe-tRNA, whereas with poly(X) there is a 2-3-fold preference for fMET-tRNA. The initiation factor IF-2, which is strictly required for the binding of N-blocked aminoacyl-tRNAs, has a higher affinity for fMET-tRNA than for AcPhe-tRNA. It is concluded that (a) interaction of the 30-S ribosomal subunit with poly(U) or poly(X) leads to IF-2-dependent binding of N-blocked aminoacyl-tRNA; (b) the initiation factor IF-2-discriminates in favour of fMET-TRNA; (c) the presence of the cognate codon discriminates in favour of the corresponding N-blocked aminoacyl-tRNA.

30S ribosomes were isolated from a kasugamycin resistant mutant of E. coli that lacks methylgroups on two adjacent adenines in 16S ribosomal RNA. These ribosomes were methylated in vitro with a purified methylating enzyme and 5-S-adenosyl-(13C-methyl)-L-methionine chloride ((13C-methyl)-SAM) as methyldonor. After in situ cleavage of the 16S ribosomal RNA by the bacteriocin cloacin DF13, the 49 nucleotide fragment from the 3'-end of the RNA was isolated. The carbon-13 nuclear magnetic resonance spectra of the fragment at various temperatures were compared with those of 6-N-dimethyladenosine (m6(2)A) and 6-N-dimethyladenylyl-(3' leads to 5')-6-N-dimethyladenosine (m6(2)Am6(2)A). The data show that the two methylated adenines, which are part of a four membered hairpin loop, show a strong tendency to be stacked in analogy to the dinucleotide m6(2)Am6(2).

Improved Parameters for Prediction of RNA Secondary Structure and Insights into why RNA forms Double Helixes.- Anomalous Conformations of RNA Constituents: 2D NMR and Calculational Studies.- NMR Studies of Base-pair Kinetics of Nucleic Acids.- Picosecond Time Domain Spectroscopy of Structure and Dynamics in Nucleic Acids.- Z-RNA: A Left-handed Double Helix.- A Better Way to Make RNA for Physical Studies.- Mg2+-inner Sphere Complexes at "Ends" and "Bends" of Polynucleotides and their Potential Role as Long Range Inducers of Conformation Changes.- Pseudoknots in RNA: A Novel Folding Principle.- Proton NMR Studies of RNA's and Related Enzymes Using Isotope Labels.- GU Base Pairs and Variable Loop in Yeast tRNAAsp.- Correlation Between Crystal and Solution Structures in tRNA. Yeast tRNAPhe and tRNAAsp the Models for Free and Messenger RNA Bound tRNAs.- The Solution Structures of RNA Fragments Determined by Nuclear Overhauser Enhancement Measurements.- Molecular Dynamics Simulation of the Anticodon Arm of Phenylalanine Transfer RNA.- Anticodon-Anticodon Interactions and tRNA Sequence Comparison: Approaches to Codon Recognition.- Studies on N Labelled 5S RNA: Assignments in the Helix V Region of 5S RNA, and in the 5S/L25 Complex.- Equilibria in Ribosomal RNA Secondary Structure.- A Comparative Analysis of Structural Dynamics in 5S rRNA.- A Domain of 23S Ribosomal RNA in Search of a function.- The Three-Dimensional Organization of Escherichia coli Ribosomal RNA.- The Extremely Small Mitochondrial Ribosomal RNAs from Trypanosomes.- Mutagenesis of Ribosomal RNA as a Method to Investigate Interactions Between rRNA, mRNA and tRNA.- Ribosomal RNA at the Decoding Site of the tRNA-Ribosome Complex.- Double Stranded RNA in the Decoding of the mRNA by the Bacterial Ribosome.- Conformational Dynamics Involved in RNA Self-Splicing.- RNA Catalyzed Lariat Formation from Yeast Mitochondrial Pre-Ribosomal RNA.- Viroids: Structure Formation and function.- Author Index.