Search PubMed⌕ Search

Biomedical subjects

H F Noller

Publications and source records attributed to H F Noller.

At least 73 records · Page 4Linked to original sources
PubMed1994

Selective perturbation of G530 of 16 S rRNA by translational miscoding agents and a streptomycin-dependence mutation in protein S12.

Previous studies have shown that a concise set of universally conserved bases in 16 S rRNA are strongly protected from attack by chemical probes when tRNA is bound specifically to the ribosomal A site. Two of these bases, A1492 and A1493, are located in the cleft of the 30 S subunit, the site of codon-anticodon interaction. A third residue, G530, is located within the highly conserved 530 stem-loop, a region that is involved in interactions with proteins S4 and S12, mutations in which perturb the translational error frequency. The 530 loop is also thought to be located at or near the site of interaction of elongation factor Tu on the 30 S subunit, a location that is distinct from the decoding site. This study monitors the response of these two A-site-related regions of 16 S rRNA to a variety of translational miscoding agents. Several of these agents, including streptomycin, neomycin and ethanol, selectively potentiate tRNA-dependent protection of residue G530 from kethoxal modification; in contrast, little change in reactivity of residues A1492 and A1493 is observed. These results are consistent with the previously demonstrated importance of G530 for A-site function and, moreover, suggest a common mechanism of action for these miscoding agents, even though they appear to have distinctly different modes of interaction with 16 S rRNA. In contrast to the miscoding agents, we find that a streptomycin-dependence (SmD) mutation in protein S12, which causes ribosomes to be hyperaccurate, antagonizes tRNA-dependent protection of G530. The possibility that 5' or 3' flanking regions of mRNA could be involved in tRNA-dependent protection of G530 was tested by using different lengths of oligo(U) to promote binding of tRNA(Phe) to the A site. The relative levels of protection of G530, A1492 and A1493 were unchanged as the size of the mRNA fragment was decreased from 16 to 6 bases in length. We conclude, therefore, that for protection of G530 to be the result of direct contact with message, it must necessarily be located directly at the decoding site; otherwise, its protection is best explained by allosteric interactions, either with mRNA, or with the codon-anticodon complex. These results are discussed in terms of a model wherein the conformation of the 530 loop is correlated with the affinity of the ribosome for elongation factor Tu.

Base Sequence↗
PubMed1994

The 530 loop of 16S rRNA: a signal to EF-Tu?

The molecular mechanism of tRNA selection has remained elusive. Recent evidence, both biochemical and genetic, suggests the involvement of 'smart' ribosomes, that can signal the elongation factor Tu by way of conformational changes in ribosomal RNA.

Models, Biological↗
PubMed1994

Constraint satisfaction techniques for modeling large complexes: application to the central domain of 16S ribosomal RNA.

Standard experimental techniques for determining the structure of small to moderately-sized molecules are difficult to apply to large macromolecular complexes. These complexes, consisting of multiple protein and/or nucleic acid components, can contain many thousands of atoms and the experimental techniques used to study them provide relatively sparse structural information with significant measurement uncertainty. Computational technologies are required to reduce the conformational search space and synthesize the data in order to produce the structures or (more usually) sets of structures compatible with the data. In this paper, we show that a method based on the constraint satisfaction paradigm produces a three-dimensional topology for the central domain of the 16S ribosomal RNA that is generally consistent with interactively built models, although differing in significant ways. The modeling incorporates information about secondary structure of the nucleic acid, neutron diffraction data about the relative positions and uncertainties of the proteins, and protection experiments indicating proximities of segments of RNA to specific protein subunits. Unlike previously proposed models, our model contains explicit information about the range of positions for each subunit that are compatible with the data. The system uses a grid search, checks distances in a direction-dependent manner, uses disjunctive distance constraints, and checks for volume overlap violations.

Animals↗
PubMed1993

Dynamics of in vitro assembly of 16 S rRNA into 30 S ribosomal subunits.

One of the important unsolved problems in the ribosome field is the molecular basis for the sequential and co-operative nature of ribosome assembly. As an approach to this problem, we have taken advantage of the temperature dependence of in vitro reconstitution and have used chemical probing methods to examine the conformation and reactivity of 16 S rRNA at successive stages during subunit assembly. One class of nucleotides displays reactivities similar to those observed in native 30 S particles when the RNA and protein are incubated in the absence of any heat step (0 degrees C effects). At 30 degrees C, where the assembly process takes 2 hours, other bases can be assigned to one of several additional kinetic classes, determined by the rate at which their chemical reactivities transit from levels observed in naked RNA to levels observed in fully assembled subunits: (1) fast (t1/2 = < 5 min at 30 degrees C); (2) slow (t1/2 = 15 to 30 min at 30 degrees C); (3) delayed slow (t1/2 = 30 to 60 min at 30 degrees C). Finally, several nucleotides display transient kinetics in their reactivities, showing increasing reactivity at early time points and becoming protected later in assembly; most of these effects correspond to residues that were previously shown to display reciprocal enhancement and protection patterns during step-wise in vitro assembly. These findings, together with our previous studies using purified individual proteins lead to the following conclusions: (1) there is a predominant 5' to 3' polarity to in vitro assembly, even though it is uncoupled from transcription; (2) portions of the central and 3' major domains fold into an active conformation only at a very late stage of assembly; (3) bases footprinted by late-assembling proteins, according to the 30 S subunit assembly map, show generally slower kinetics than residues footprinted by proteins that bind early in the assembly map, providing direct evidence for the sequential nature of the in vitro assembly process; (4) most proteins are associated with nucleotides that fall into more than one kinetic class, suggesting that assembly proceeds through multiple pathways, or that individual proteins interact sequentially with different regions of the RNA.

Cold Temperature↗
PubMed1993

Footprinting the sites of interaction of antibiotics with catalytic group I intron RNA.

Aminoglycoside inhibitors of translation have been shown previously to inhibit in vitro self-splicing by group I introns. Chemical probing of the phage T4-derived sunY intron shows that neomycin, streptomycin, and related antibiotics protected the N-7 position of G96, a universally conserved guanine in the binding site for the guanosine cofactor in the splicing reaction. The antibiotics also disrupted structural contacts that have been proposed to bring the 5' cleavage site of the intron into proximity to the catalytic core. In contrast, the strictly competitive inhibitors deoxyguanosine and arginine protected only the N-7 position of G96. Parallels between these results and previously observed protection of 16S ribosomal RNA by aminoglycosides raise the possibility that group I intron splicing and transfer RNA selection by ribosomes involve similar RNA structural motifs.

Aminoglycosides↗
PubMed1993

Evidence for functional interaction between elongation factor Tu and 16S ribosomal RNA.

Translation of the genetic code requires the accurate selection of elongation factor (EF)-Tu.GTP.tRNA ternary complexes at the ribosomal acceptor site, or A site. Several independent lines of evidence have implicated the universally conserved 530 loop of 16S rRNA in this process; yet its precise role has not been identified. Using an allele-specific chemical probing strategy, we have examined the functional defect caused by a dominant lethal G-->A substitution at position 530. We find that mutant ribosomes are impaired in EF-Tu-dependent binding of aminoacyl-tRNA in vitro; in contrast, nonenzymatic binding of tRNA to the A and P sites is unaffected, indicating that the defect involves an EF-Tu-related function rather than tRNA-ribosome interactions per se. In vivo, the mutant ribosomes are found in polysomes at low levels and contain reduced amounts of A-site-bound tRNA, but normal levels of P-site tRNA, in agreement with the in vitro results; thus the dominant lethal phenotype of mutations at G530 can be explained by impaired interaction of mutant ribosomes with ternary complex. These results provide evidence for a newly defined function of 16S rRNA--namely, modulation of EF-Tu activity during translation.

Alleles↗
PubMed1993

Allele-specific structure probing of plasmid-derived 16S ribosomal RNA from Escherichia coli.

Biochemical analysis of Escherichia coli ribosomes containing mutant 16S or 23S (r)ribosomal RNAs, produced via cloned rDNA genes on multicopy plasmids, has been hindered by the background of wild-type (wt) ribosomes containing host-derived rRNA. Here, we describe a method for the construction of unique priming sites in 16S rRNA that allow allele-specific structure probing of ribosomes containing plasmid-encoded RNA. Phenotypically silent mutations, designed to mimic related eubacterial sequences, have been introduced into four phylogenetically variable regions in the 16S rDNA gene that allow inspection of several 16S rRNA functional sites. When oligodeoxyribonucleotides complementary to these altered sequences are used to prime cDNA synthesis in primer extension reactions using reverse transcriptase, only plasmid-derived 16S rRNA is used as a template, thus rendering the wt background invisible. Unexpectedly, we were unable to introduce silent mutations into one nonconserved helix in 16S rRNA, suggesting that constraints in addition to Watson-Crick pairing are important in this region.

Alleles↗
PubMed1993

Methylation interference experiments identify bases that are essential for distinct catalytic functions of a group I ribozyme.

Methylation interference experiments reveal bases involved in three different catalytic functions of the T4-phage derived sunY self-splicing intron. RNA molecules methylated at the N-7 position of the guanine at the cofactor binding site are inactive in cofactor-dependent splicing and 3' splice-site hydrolysis. In contrast, 5' splice-site hydrolysis occurs despite methylation at this position. Specific adenines that have been implicated in docking of the P1 stem to the catalytic core are shown to be important for cofactor-dependent splicing and essential for 5' splice-site hydrolysis. Similarly, methylation of bases in the P9.0 stem, as well as C56 in J5/4, interferes with 3' splice-site hydrolysis and with the splicing reaction. All of the bases identified as important for the overall splicing reaction are also identified as essential for either the 5' or 3' splice-site hydrolysis reactions, and vice versa. It is inferred that the bases implicated in 5' and 3' splice-site hydrolysis are involved in specific interactions of the 5' and 3' splice site, respectively, with the catalytic core.

Bacteriophage T4↗
PubMed1993

tRNA-rRNA interactions and peptidyl transferase.

The extent to which ribosomal RNA is directly involved in the function of ribosomes has important implications for both the mechanism of translation and the molecular origins of life. Detailed evidence has accumulated that places the anticodon and acceptor ends of tRNA in close proximity to conserved features of rRNA in the ribosome. Recent studies are providing evidence that these features are important for ribosomal function.

Base Sequence↗
PubMed1993

A cold-sensitive mutation in 16S rRNA provides evidence for helical switching in ribosome assembly.

A C-->U substitution at position 23 of 16S rRNA confers a dominant, cold-sensitive phenotype. The mutation changes the G11-C23 base pair of the 5' terminal pseudoknot helix to a G-U pair, which is predicted to cause significant weakening of the helix. Ribosomes containing mutant RNA are impaired in assembly and function at low temperature. Cells expressing the C23U mutation have decreased polysome levels and accumulate free 30S and 50S subunits and particles that resemble those previously observed in cold-sensitive alleles of ribosomal protein S5 and in in vitro reconstitution of 30S subunits carried out at low temperature. Three second-site suppressor mutations suggest that cold sensitivity is caused by competition between the 5' helix and an alternative helix formed by base-pairing of the upstream precursor sequence with one strand of the mature helix. Cold sensitivity appears to be relieved by destabilization of the competing precursor helix relative to the mature helix.

Base Sequence↗
PubMed1992

Cleavage of tRNA by Fe(II)-bleomycin.

We have investigated the action of the chemotherapeutic agent Fe(II)-bleomycin on yeast tRNA(Phe), an RNA of known three-dimensional structure. In the absence of Mg2+ ions, the RNA is cleaved preferentially at two major positions, A31 and G53, both of which are located at the terminal base pairs of hairpin loops, and coincide with the location of tight Mg2+ binding sites. A fragment of the tRNA (residues 47-76) containing the T stem-loop is also cleaved specifically at G53. Cleavage of both the intact tRNA and the tRNA fragment is abolished in the presence of physiological concentrations of Mg2+ (> 0.5 mM). Since Fe(II) is not displaced from bleomycin under these conditions, we infer that tight binding of Mg2+ to tRNA excludes productive interactions between Fe(II)-bleomycin and the RNA. These results also show that loss of cleavage is not due to Mg(2+)-dependent formation of tertiary interactions between the D and T loops. In contrast, cleavage of synthetic DNA analogs of the anticodon and T stem-loops is not detectably inhibited by Mg2+, even at concentrations as high as 50 mM. In addition, the site specificities observed in cleavage of RNA and DNA differ significantly. From these results, and from similar findings with other representative RNA molecules, we suggest that the cleavage of RNA by Fe(II)-bleomycin is unlikely to be important for its therapeutic action.

Base Sequence↗
PubMed1992

Hydroxyl radical cleavage of tRNA in the ribosomal P site.

Hydroxyl radical is a useful probe of the accessibility of the sugar moiety of nucleic acids to solvent. Here we compare the accessibility of free and ribosome-bound yeast tRNA(Phe), Escherichia coli tRNA(Phe), and E. coli tRNA(Leu2) to attack by hydroxyl radicals generated from Fe(2+)-EDTA. When bound to the P site of 30S ribosomal subunits, a discrete region, corresponding almost precisely to the anticodon stem-loop, is strongly protected; weaker protection is observed in the 3' strand of the D stem and in the variable loop. The protected nucleotides constitute a well-defined substructure, corresponding to the lower half of the anticodon-D loop coaxial arm of the tRNA crystal structure. This result suggests that the 30S P site contains a pocket that becomes inaccessible to the Fe(2+)-EDTA complex when tRNA is bound, whose minimum dimensions can be inferred from the boundaries of the protected region of tRNA. When bound to the P site of 70S ribosomes, the entire tRNA backbone becomes inaccessible to hydroxyl radicals. Since previous studies have shown that virtually the entire footprint of a P-site tRNA on 16S and 23S rRNAs is mimicked by the extremities of the tRNA (the anticodon stem-loop plus the 3'-terminal aminoacyl-pentanucleotide), protection of the entire tRNA was unexpected. We conclude that protection of the elbow of tRNA is due either to interactions with ribosomal proteins or to enclosure in an inaccessible site formed by association of the two ribosomal subunits.

Base Sequence↗
PubMed1992

Unusual resistance of peptidyl transferase to protein extraction procedures.

Peptidyl transferase, the ribosomal activity responsible for catalysis of peptide bond formation, is resistant to vigorous procedures that are conventionally employed to remove proteins from protein-nucleic acid complexes. When the "fragment reaction" was used as a model assay for peptide bond formation, Escherichia coli ribosomes or 50S subunits retained 20 to 40 percent activity after extensive treatment with proteinase K and SDS, but lost activity after extraction with phenol or exposure to EDTA. Ribosomes from the thermophilic eubacterium Thermus aquaticus remained more than 80 percent active after treatment with proteinase K and SDS, which was followed by vigorous extraction with phenol. This activity is attributable to peptidyl transferase, as judged by specific inhibition by the peptidyl transferase-specific antibiotics chloramphenicol and carbomycin. In contrast, activity is abolished by treatment with ribonuclease T1. These findings support the possibility that 23S ribosomal RNA participates in the peptidyl transferase function.

Electrophoresis, Polyacrylamide Gel↗
PubMed1992

Aminoacyl esterase activity of the Tetrahymena ribozyme.

Several classes of ribozymes (catalytic RNA's) catalyze reactions at phosphorus centers, but apparently no reaction at a carbon center has been demonstrated. The active site of the Tetrahymena ribozyme was engineered to bind an oligonucleotide derived from the 3' end of N-formyl-methionyl-tRNA(fMet). This ribozyme catalyzes the hydrolysis of the aminoacyl ester bond to a modest extent, 5 to 15 times greater than the uncatalyzed rate. Catalysis involves binding of the oligonucleotide to the internal guide sequence of the ribozyme and requires Mg2+ and sequence elements of the catalytic core. The ability of RNA to catalyze reactions with aminoacyl esters expands the catalytic versatility of RNA and suggests that the first aminoacyl tRNA synthetase could have been an RNA molecule.

Animals↗
PubMed1991

A single base substitution in 16S ribosomal RNA suppresses streptomycin dependence and increases the frequency of translational errors.

A C to U substitution at position 1469 of 16S ribosomal RNA (rRNA) from Escherichia coli suppresses streptomycin dependence and causes increased translational error frequencies. Strains containing the rpsL252 or StrM287 streptomycin-dependent alleles are able to grow in the absence of streptomycin when transformed with plasmids containing the U1469 mutation in 16S rRNA. Ribosomes containing wild-type proteins and U1469 mutant 16S rRNA misincorporate leucine in vitro at elevated levels, comparable to that of some typical S4 ram ribosomes. These results provide additional support for the participation of 16S rRNA in maintaining translational accuracy.

Base Sequence↗
PubMed1991

Sites of interaction of the CCA end of peptidyl-tRNA with 23S rRNA.

Oligonucleotide fragments derived from the 3' CCA terminus of acylated tRNA, such as CACCA-(AcPhe), UACCA-(AcLeu), and CAACCA-(fMet), bind specifically to ribosomes in the presence of sparsomycin and methanol [Monro, R. E., Celma, M. L. & Vazquez, D. (1969) Nature (London) 222, 356-358]. All three oligonucleotides protect a characteristic set of bases in 23S rRNA from chemical probes: G2252, G2253, A2439, A2451, U2506, and U2585. A2602 shows enhanced reactivity. These account for most of the same bases that are protected when peptidyl-tRNA analogues such as AcPhe-tRNA are bound to the ribosomal P site, and correspond precisely to those bases whose protection is abolished by removal of the 3'-CA end of tRNA. We conclude that most of the observed interactions between tRNA and 23S rRNA in the 50S ribosomal P site involve the conserved CCA terminus of tRNA. Sparsomycin may inhibit protein synthesis by stabilizing interaction between the peptidyl-CCA and the 23S P site, preventing formation of the intermediate A/P hybrid state.

Base Sequence↗