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H F Noller

Publications and source records attributed to H F Noller.

At least 127 records · Page 7Linked to original sources
PubMed1984

The conserved 5 S rRNA complement to tRNA is not required for translation of natural mRNA.

We have tested a putative base-paired interaction between the conserved GT psi C sequence of tRNA and the conserved GAAC47 sequence of 5 S ribosomal RNA by in vitro protein synthesis using ribosomes containing deletions in this region of 5 S rRNA. Ribosomes reconstituted with 5 S rRNA possessing a single break between residues 41 and 42, deletion of residues 42-46, or deletion of residues 42-52 were tested for their ability to translate phage MS2 RNA. Initiator tRNA binding, aminoacyl-tRNA binding, ppGpp synthesis, and miscoding were also tested. All of the measured functions could be carried out by ribosomes carrying the deleted 5 S rRNAs. The sizes and relative amounts of the polypeptides synthesized by MS2 RNA-programmed ribosomes were identical whether or not the 5 S RNA contained deletions. Aminoacyl-tRNA binding and miscoding were essentially unaffected. Significant reduction in ApUpG (but not poly(A,U,G) or MS2 RNA)-directed fMet-tRNA binding and ppGpp synthesis were observed, particularly in the case of the larger (residues 42-52) deletion. We conclude that if tRNA and 5 S rRNA interact in this fashion, it is not an obligatory step in protein synthesis.

Base Composition↗
PubMed1984

The nucleotide sequence of a rat 18 S ribosomal ribonucleic acid gene and a proposal for the secondary structure of 18 S ribosomal ribonucleic acid.

The nucleotide sequence of a rat 18 S rRNA gene was determined. The 18 S rRNA encoded in the gene contains 1874 nucleotides, and the molecular weight estimated from the sequence is 6.09 X 10(5). The sequences of rat and Xenopus laevis 18 S rRNAs are very similar; the only differences of consequence are the insertions between nucleotides 197 and 206 and between 258 and 279 of the rat nucleic acid of sequences rich in guanine and cytosine. A proposal is presented for the secondary structure of rat 18 S rRNA based on a comparison with the sequences of 17 other small ribosomal subunit RNAs. While the primary sequences of rat and eubacterial RNAs are different, the deduced secondary structures are remarkably similar.

Animals↗
PubMed1984

Identification of a site on 23S ribosomal RNA located at the peptidyl transferase center.

3-(4'-Benzoylphenyl)propionyl[3H] Phe-tRNA bound to the peptidyl site of the ribosome is photo-crosslinked exclusively to 23S RNA on irradiation at 320 nm. The site of reaction has been identified both by hybridization and primer-extension experiments as uridine-2584 and uridine-2585, located within the central loop of domain V according to the secondary structure model of 23S RNA. The fact that the covalently crosslinked tRNA retains its ability to form a peptide bond, together with the proximity of this site to the position of several mutations leading to chloramphenicol or erythromycin resistance strongly argue that this region of the 23S-like rRNAs is an integral component of the peptidyl transferase site. On the basis of these results, and from comparative analysis of the 16 available large subunit rRNA sequences, we propose a model for the functional organization of the peptidyl transferase site involving interaction of domains II and V of 23S rRNA.

Acyltransferases↗
PubMed1984

Complete nucleotide sequence of a 23S ribosomal RNA gene from Bacillus stearothermophilus.

A 23S ribosomal (rRNA) gene from Bacillus stearothermophilus has been cloned in pBR322, and its nucleotide sequence determined. The corresponding mature 23S rRNA is predicted to contain 2928 nucleotides. We compare the primary and secondary structures of 23S rRNA from Escherichia coli and B. stearothermophilus, and discuss their potential contributions to thermal stability.

Base Sequence↗
PubMed1983

Ribonucleic acid-protein cross-linking within the intact Escherichia coli ribosome, utilizing ethylene glycol bis[3-(2-ketobutyraldehyde) ether], a reversible, bifunctional reagent: synthesis and cross-linking within 30S and 50S subunits.

We have used the reversible, bifunctional reagent ethylene glycol bis[3-(2-ketobutyraldehyde) ether] to cross-link RNA to protein within intact ribosomal subunits from Escherichia coli. Here we describe the synthesis of this compound (termed bikethoxal) and demonstrate its ability to form covalent attachments between RNA and protein in the 5S RNA-L18 complex and within 30S and 50S ribosomal subunits. The reagent is a symmetrical dicarbonyl compound and reacts with guanine in single-stranded RNA and with arginine in protein. RNA-protein cross-links generated with this reagent are stable, as demonstrated by the comigration of 35S-labeled ribosomal proteins with ribosomal RNA on neutrally buffered sodium dodecyl sulfate (SDS)-agarose gels. However, the cross-linked product is unstable in mildly basic conditions, allowing the identification of the linked macromolecules by conventional techniques. The reagent is potentially capable of cross-linking any combination of single-stranded RNA, single-stranded DNA, or protein; it should prove a useful probe of the RNA-protein proximities within the E. coli ribosome, since the SDS-agarose gel system we describe provides a rapid method of optimizing this RNA--protein cross-linking reaction.

Aldehydes↗
PubMed1983

Ribonucleic acid-protein cross-linking within the intact Escherichia coli ribosome, utilizing ethylene glycol bis[3-(2-ketobutyraldehyde) ether], a reversible, bifunctional reagent: identification of 30S proteins.

To obtain detailed topographical information concerning the spatial arrangement of the multitude of ribosomal proteins with respect to specific sequences in the three RNA chains of intact ribosomes, a reagent capable of covalently and reversibly joining RNA to protein has been synthesized [Brewer, L.A., Goelz, S., & Noller, H. F. (1983) Biochemistry (preceding paper in this issue)]. This compound, ethylene glycol bis[3-(2-ketobutyraldehyde) ether] which we term "bikethoxal", possesses two reactive ends similar to kethoxal. Accordingly, it reacts selectively with guanine in single-stranded regions of nucleic acid and with arginine in protein. The cross-linking is reversible in that the arginine- and guanine-bikethoxal linkage can be disrupted by treatment with mild base, allowing identification of the linked RNA and protein components by standard techniques. Further, since the sites of kethoxal modification within the RNA sequences of intact subunits are known, the task of identifying the components of individual ribonucleoprotein complexes should be considerably simplified. About 15% of the ribosomal protein was covalently cross-linked to 16S RNA by bikethoxal under our standard reaction conditions, as monitored by comigration of 35S-labeled protein with RNA on Sepharose 4B in urea. Cross-linked 30S proteins were subsequently removed from 16S RNA by treatment with T1 ribonuclease and/or mild base cleavage of the reagent and were identified by two-dimensional polyacrylamide gel electrophoresis. The major 30S proteins found in cross-linked complexes are S4, S5, S6, S7, S8, S9 (S11), S16, and S18. The minor ones are S2, S3, S12, S13, S14, S15, and S17.

Aldehydes↗
PubMed1983

Identification of sites of 4'-(hydroxymethyl)-4,5',8-trimethylpsoralen cross-linking in Escherichia coli 23S ribosomal ribonucleic acid.

The reagent 4'-(hydroxymethyl)-4,5',8-trimethylpsoralen (HMT) was used to cross-link 23S rRNA from Escherichia coli under 50S ribosomal subunit reconstitution conditions. Following partial digestion of the RNA with ribonuclease T1, two-dimensional diagonal electrophoresis in denaturing polyacrylamide gels was used to isolate fragments derived from the cross-linked sites. These fragments were analyzed by digestion with ribonucleases T1 and A and their positions in the 23S RNA sequence identified. Fragment a1 (positions 1325-1426) is cross-linked to a2 (positions 1574-1623); fragment b1 (positions 1700-1731) is cross-linked to b2 (positions 1732-1753); and a cross-link is formed within fragment c (or c') (positions 863-916). In the latter case, the cross-link was located precisely, linking residues C867 and U913. All three HMT-mediated cross-links are consistent with a proposed secondary structure model for 23S RNA [Noller, H. F., Kop, J., Wheaton, V., Brosius, J., Gutell, R. R., Kopylov, A. M., Dohme, F., Herr, W., Stahl, D. A., Gupta, R., & Woese, C. R. (1981) Nucleic Acids Res. 9, 6167-6189].

Base Sequence↗
PubMed1983

Protection of ribosomal RNA from kethoxal in polyribosomes. Implication of specific sites in ribosome function.

In an attempt to probe the topography of 5 S, 16 S and 23 S RNAs in a functionally engaged ribosome, polysomes were probed using the structure-sensitive, guanine-specific reagent kethoxal. Reactivities of guanine residues at 38 specific ribosomal RNA sites in polysomes were compared with their corresponding reactivities in vacant 70 S ribosomes. No polysome-specific protection was seen for 5 S RNA. In 16 S RNA, positions 530, 693 or 1079, 966, 1338 and 1517 showed protection in polysomes; all of these sites have highly conserved primary and secondary structures, and include several methylated nucleotides. In 23 S RNA, polysome protection is seen at positions 277, 1071, 1475 or 2112, 2116 and 2751. We attribute polysome-specific protection either to direct contact of transfer RNA and/or messenger RNA with the protected sites or to tRNA and/or mRNA-induced changes in ribosome conformation involving the protected sites.

Aldehydes↗
PubMed1982

Identification of a site of psoralen crosslinking in E. coli 16S ribosomal RNA.

We have developed a 2-dimensional gel method for identification of RNA sequences crosslinked by the intercalative drug 4'-hydroxymethyl-4,5',8-trimethylpsoralen (HMT). This method is being used to localize such sites in E. coli ribosomal RNA. We report here the identification of a site for HMT crosslinking within positions 434 and 497 of 16S rRNA. We suggest a likely site for HMT intercalation, in which residues U548 and U473 become crosslinked via the drug.

Base Sequence↗
PubMed1981

Secondary structure model for 23S ribosomal RNA.

A secondary structure model for 23S ribosomal RNA has been constructed on the basis of comparative sequence data, including the complete sequences from E. coli. Bacillus stearothermophilis, human and mouse mitochondria and several partial sequences. The model has been tested extensively with single strand-specific chemical and enzymatic probes. Long range base-paired interactions organize the molecule into six major structural domains containing over 100 individual helices in all. Regions containing the sites of interaction with several ribosomal proteins and 5S RNA have been located. Segments of the 23S RNA structure corresponding to eucaryotic 5.8S and 25 RNA have been identified, and base paired interactions in the model suggest how they are attached to 28S RNA. Functionally important regions, including possible sites of contact with 30S ribosomal subunits, the peptidyl transferase center and locations of intervening sequences in various organisms are discussed. Models for molecular 'switching' of RNA molecules based on coaxial stacking of helices are presented, including a scheme for tRNA-23S RNA interaction.

Animals↗
PubMed1981

Secondary structure of 16S ribosomal RNA.

A secondary structure model for 16S ribosomal RNA which is based on available chemical, enzymatic, and comparative sequence data shows good agreement between constraints dictated by the model and a wide variety of experimental observations. The four major structural domains created by the base-pairing scheme correspond closely to RNA fragments isolated after nuclease digestion in the presence of bound ribosomal proteins. Functionally important sites appear to be located in unpaired regions and are phylogenetically highly conserved.

Base Sequence↗
PubMed1981

Ribonucleic acid-protein cross-linking in Escherichia coli ribosomes: (4-azidophenyl)glyoxal, a novel heterobifunctional reagent.

We have used the heterobifunctional reagent (4-azidophenyl)glyoxal (APG) to cross-link RNA to protein in Escherichia coli 30S ribosomal subunits. Synthesis and characterization of the reagent are described. Like other dicarbonyl reagents (e.g., kethoxal), APG reacts specifically with guanosine among the four ribonucleosides. The azido group in APG can be photolyzed with UV light (lambda greater than 300 nm), yielding an unstable nitrene which is potentially reactive with many groups in proteins and nucleic acids. Conditions for APG modification of guanylic acid residues in 30S subunits are described; photolysis of bound APG results in cross-linking of approximately 5% of the total 30S proteins to 16S RNA. A specific subset of the 30S proteins is cross-linked to 16S RNA by APG.

Aldehydes↗