Functional 30S ribosomes were reconstructed from total Escherichia coli 30S ribosomal proteins and 16S ribosomal RNA synthesized in vitro by T7 RNA polymerase. Up to 700 mol of RNA/mol of template could be obtained. The transcript lacked all ten normally modified bases and had three additional 5' G residues, an A----G change at position 2, and, in 22% of the molecules, one or two extra 3' residues. The synthetic 16S RNA could be assembled into a particle that cosedimented with authentic 30S and was indistinguishable from 30S by electron microscopy. When supplemented with the 50S subunit, the particles bound tRNA to the 70S P site in a codon- and Mg2+-dependent manner. The specific binding activity was 94% that of particles reconstituted with natural rRNA and 52% that of native 30S. Cross-linking to P site bound tRNA was also preserved. Changing C-1400, the residue known to be close to the anticodon of P site bound tRNA, to A had little effect on reconstitution, but the C----G substitution caused a marked inhibition of assembly. tRNA could bind to both reconstituted mutants, but cross-linking was greatly reduced. These results show that none of the modified bases of 16S RNA are essential for P site binding and that position 1400 may be more important for ribosome assembly than for tRNA binding. Base-specific in vitro mutagenesis can now be used to explore in detail the functional properties of individual residues in ribosomal RNA.

An enhanced lateral view of the 40S ribosomal subunit of HeLa cells has been obtained by computer averaging of single particles visualized in the electron microscope. Application of crystallographic criteria to independent averages shows that the reproducibility of the result is comparable to that obtained for thin, stained protein crystals by conventional Fourier filtration methods.

No abstract

Proton magnetic resonance, circular dichroism and other studies of whole and cleaved calf thymus histone H1 (formerly F1) reveal the presence of specific folded structures in the region approximately from residue 40-115. Ionic, hydrogen-bond and hydrophobic interactions all appear to contribute to the stability of the structure, which is predicted to contain a-helices in regions 42-55 and 58-75. No evidence was found for 8-structures, either inter or intramolecular, or for any structure formation outside the region 40-115. At 18 "C and a protein concentration of 2 mM the first-order exchange rate between random-coil and structured forms is slower than 80 s-'; at 40 "C the exchange rate is faster than 330 s-The very-lysine-rich histone H 1 (formerly called F1 or I; the nomenclature used in this paper is taken from [I]), exhibits several features which distinguish it from the other histone fractions found in eukaryote chromatin. It has the highest molecular weight (approx. 23000) and has a very high lysine : arginine ratio of over 15 : 1, varying up to 21 : 1 for some subfractions.Over 25 % of the molecule consists of lysine residues, but despite this very basic nature histone H1 is the fraction most easily removed from chromatin on increasing the ionic strength of the solution. Histone H1 has been implicated in the condensation of chromatin in two ways : increase in ionic strength of a chromatin gel in the region below that required to remove histone H1 (0.1-0.3 M NaC1) causes a ten-fold physical contraction of the gel which is dependent on the Abbreviations. NMR, nuclear magnetic resonance; CD, circular dichroism. presence of histone H1 [2]; and in the true slime mould P. polycephalum the very-lysine-rich histone H1 undergoes a peak of phosphorylation late in G2 phase at a point in the cell cycle which corresponds to chromosome condensation [3]. This latter observation has led to the proposal that phosphorylation of histone H1 may be part of a mitotic trigger mechanism [4,5]. Furthermore, a histone Hl/DNA reconstituted complex also exhibits physical condensation at the same ionic strengths as those required for chromatin. This effect and that of histone H1 in chromatin gel will be described in the succeeding paper of this series.Mammalian histone H1 has been subjected to several sequence determinations, which reveal some sequence microheterogeneity which is both species and tissue specific [7,8]. The experiments described in this paper were carried out using unfractionated calf thyEur. J. Biochem. 52 (1975)

High resolution nuclear magnetic resonance spectroscopy is used to study conformational changes in histone fractions F2a1 and FI when the ionic strength of aqueous solutions is raised.Increasing line widths of certain resonance peaks, in particular those of apolar and aromatic amino acids, together with sequence data lead to the conclusion that the C-terminal half of F2a1 and a central portion of F I are involved in the conformational changes. The proportion of amino acid residues incorporated into secondary structure (as indicated by optical rotatory dispersion) is less than that involved in the conformational changes indicated by the nuclear magnetic resonance results. Intermolecular interactions are therefore postulated to explain this difference and these are specific in as much as they involve only a part of the histone molecule and include the regions of the chain having high potential for secondary structure formation.The complete amino acid sequence determination of the glycine-arginine rich histone fraction F2a1 from calf thymus by Smith and his collaborators [2] has revealed a striking feature, namely that certain amino acids are by no means evenly distributed along the polypeptide chain. This possibility was suggested some while ago by Phillips [3] from the results of tryptic digests of these histones which yielded insoluble 'cores' containing a high number of apolar amino acids and showed the average distance between basic residues to vary between 0 and 8. A similar irregular distribution of amino acids has been observed in the lysine rich histone F1 from the compositions and alignment of three sets of peptides [4].The distributions of amino acids in the amino and carboxyl halves of F1 and F2a1 together with the amino acid compositions of some enzymes are shown in Table I . The differences between the amino and carboxyl halves of the histones are strikingly demonstrated, the amino-terminal half of F2a, being very similar in character to the carboxyl-terminal half of F1 ; the main difference being that glycine in FZa, is replaced by proline in F1, both presumably acting strongly against helix formation. It has been suggested [1,4] that these basic ends of F1 and FZa, are the primary sites of interaction with DNA. The carboxyl-terminal half of F2a1 is very similar in composition to the amino half of F1 except for the very much smaller number of aromatic residues in FI.

We have partially purified two 16S rRNA-specific methyltransferases, one of which forms m2G966 (m2G MT), while the other one makes m5C967 (m5C MT). The m2G MT uses unmethylated 30S subunits as a substrate, but not free unmethylated 16S rRNA, while the m5C MT functions reciprocally, using free rRNA but not 30S subunits (Nègre, D., Weitzmann, C. and Ofengand, J. (1990) UCLA Symposium: Nucleic Acid Methylation (Alan Liss, New York), pp. 1-17). We have now determined the basis for this unusual inverse specificity at adjacent nucleotides. Binding of ribosomal proteins S7, S9, and S19 to unmodified 16S rRNA individually and in all possible combinations showed that S7 plus S19 were sufficient to block methylation by the m5C MT, while simultaneously inducing methylation by the m2G MT. A purified complex containing stoichiometric amounts of proteins S7, S9, and S19 bound to 16S rRNA was isolated and shown to possess the same methylation properties as 30S subunits, that is, the ability to be methylated by the m2G MT but not by the m5C MT. Since binding of S19 requires prior binding of S7, which had no effect on methylation when bound alone, we attribute the switch in methylase specificity solely to the presence of RNA-bound S19. Single-omission reconstitution of 30S subunits deficient in S19 resulted in particles that could not be efficiently methylated by either enzyme. Thus while binding of S19 is both necessary and sufficient to convert 16S rRNA into a substrate of the m2G MT, binding of either S19 alone or some other protein or combination of proteins to the 16S rRNA can abolish activity of the m5C MT. Binding of S19 to 16S rRNA is known to cause local conformational changes in the 960-975 stem-loop structure surrounding the two methylated nucleotides (Powers, T., Changchien, L.-M., Craven, G. and Noller, H.F. (1988) J. Mol. Biol. 200, 309-319). Our results show that the two ribosomal RNA MTs studied in this work are exquisitely sensitive to this small but nevertheless functionally important structural change.

No abstract

Conformational changes in histone H2A (ALK, F2A2, IIbl) as a function of ionic strength and pH have been followed using high resolution nuclear magnetic resonance (NMR), circular dichroism (CD), and infrared (ir). While change in pH from 3 to 7 (no added salt) causes little structural change, added salt induces the formation of both alpha helix (28 percent maximum) and intermolecular associates in the region of the molecule between 25 and 113. No beta structure was observed at high salt. By the use of different salts it was shown that the structural changes were due largely to nonspecific counterion screening by the added anion. Comparison of observed with simulated NMR spectra has led to the proposal that an ionic strength dependent equilibrium exists between largely unstructured coil molecules and fully structured and aggregated molecules. NMR spectra of H2A obtained in the presence of DNA showed that both the N- and C-terminal regions bind to DNA, i.e., not the portion of the chain that is involved in interhistone interactions.