Biomedical subjects

H F Noller

Publications and source records attributed to H F Noller.

At least 55 records · Page 3Linked to original sources

PubMed1997

Ribosomes and translation.

The ribosome is a large multifunctional complex composed of both RNA and proteins. Biophysical methods are yielding low-resolution structures of the overall architecture of ribosomes, and high-resolution structures of individual proteins and segments of rRNA. Accumulating evidence suggests that the ribosomal RNAs play central roles in the critical ribosomal functions of tRNA selection and binding, translocation, and peptidyl transferase. Biochemical and genetic approaches have identified specific functional interactions involving conserved nucleotides in 16S and 23S rRNA. The results obtained by these quite different approaches have begun to converge and promise to yield an unprecedented view of the mechanism of translation in the coming years.

Animals↗

PubMed1997

Identification of 2'-hydroxyl groups required for interaction of a tRNA anticodon stem-loop region with the ribosome.

Synthetic RNA stem loops corresponding to positions 28-42 in the anticodon region of tRNA(Phe) bind efficiently in an mRNA-dependent manner to ribosomes, whereas those made from DNA do not. In order to identify the positions where ribose is required, the anticodon stem-loop region of tRNA(Phe) (Escherichia coli) was synthesized chemically using a mixture of 2'-hydroxyl- and 2'-deoxynucleotide phosphoramidites. Oligonucleotides whose ribose composition allowed binding were retained selectively on nitrocellulose filters via binding to 30S ribosomal subunits. The binding-competent oligonucleotides were submitted to partial alkaline hydrolysis to identify the positions that were enriched for ribose. Quantification revealed a strong preference for a 2'-hydroxyl group at position U33. This was shown directly by the 50-fold lower binding affinity of a stem loop containing a single deoxyribose at position U33. Similarly, defective binding of the corresponding U33-2'-O-methyl-substituted stem-loop RNA suggests that absence of the 2'-hydroxyl group, rather than an altered sugar pucker, is responsible. Stem-loop oligoribonucleotides from different tRNAs with U33-deoxy substitutions showed similar, although quantitatively different effects, suggesting that intramolecular rather than tRNA-ribosome interactions are affected. Because the 2'-hydroxyl group of U33 was shown to be a major determinant of the U-turn of the anticodon loop in the crystal structure of tRNA(Phe) in yeast, our finding might indicate that the U-turn conformation in the anticodon loop is required and/or maintained when the tRNA is bound to the ribosomal P site.

Anticodon↗

PubMed1996

Site-directed hydroxyl radical probing of the rRNA neighborhood of ribosomal protein S5.

Cysteine residues were introduced into three different positions distributed on the surface of ribosomal protein S5, to serve as targets for derivatization with an Fe(II)-ethyl-enediaminetetraacetic acid linker. Hydroxyl radicals generated locally from the tethered Fe(II) in intermediate ribonucleoprotein particles or in 30S ribosomal subunits reconstituted from derivatized S5 caused cleavage of the RNA, resulting in characteristically different cleavage patterns for the three different tethering positions. These findings provide constraints for the three-dimensional folding of 16S ribosomal RNA (rRNA) and for the orientation of S5 in the 30S subunit, and they further suggest that antibiotic resistance and accuracy mutations in S5 may involve perturbation of 16S rRNA.

Anti-Bacterial Agents↗

PubMed1996

Mapping the rRNA neighborhood of the acceptor end of tRNA in the ribosome.

In order to map the rRNA environment of the acceptor end of tRNA in th e ribosome, hydroxyl radicals were generated in situ from Fe(II) attached via an EDTA linker to the 5' end of tRNA. Nucleotides in rRNA cleaved by the radicals were identified by primer extension, and assigned to the ribosomal A, P and E sites by standard criteria. In the A site, cleavages were found in the 2555-2573 region of 23S rRNA, around bases previously shown to be protected by A site tRNA, and in the alpha-sarcin loop, the site of interaction of elongation factors EF-Tu and EF-G. P site cleavages occurred in the 2250 loop, where a base pair is made with C74 of tRNA; and around the 2493 region in domain V. Interestingly, two clusters of nucleotides in 23S rRNA are accessible to both A site and P site tRNA probes. The first cluster is in the 1940-1965 region of domain IV, around the site of affinity labeling by the 3' end of tRNA, and the second cluster is around the bulged adenosine A2602, whose accessibility to chemical probes is enhanced by P site tRNA and decreased by A site tRNA. From the E site, cleavages occur in the 2390-2440 region, surrounding C2394, a base protected from dimethyl sulfate by E site tRNA, and in the phylogenetically variable stem at positions 1860/1880 of domain IV. Unexpectedly, no cleavages were detected in the central loop of domain V of 23S rRNA.

Binding Sites↗

PubMed1996

Directed hydroxyl radical probing of the rRNA neighborhood of ribosomal protein S13 using tethered Fe(II).

Directed hydroxyl radical probing was used to probe the rRNA neighborhood around protein S13 in the 30S ribosomal subunit. The unique cysteine at position 84 of S13 served as a tethering site for attachment of Fe(II)-1-(p-bromoacetamidobenzyl)-EDTA. Derivatized S13 (Fe-C84-S13) was then assembled into 30S ribosomal subunits by in vitro reconstitution with 16S rRNA and a mixture of the remaining 30S subunit proteins. Hydroxyl radicals generated from the tethered Fe(II) resulted in cleavage of the RNA backbone in two localized regions of the 3' major domain of 16S rRNA. One region spans nt 1308-1333 and is close to a site previously crosslinked to S13. A second set of cleavages is found in the 950/1230 helix. Both regions have been implicated in binding of S13 by previous chemical footprinting studies using base-specific chemical probes and solution-based hydroxyl radical probing. These results place both regions of 16S rRNA in proximity to position C84 of S13 in the three-dimensional structure of the 30S ribosomal subunit.

Bacterial Proteins↗

PubMed1996

Computational methods for defining the allowed conformational space of 16S rRNA based on chemical footprinting data.

Structural models for 16S ribosomal RNA have been proposed based on combinations of crosslinking, chemical protection, shape, and phylogenetic evidence. These models have been based for the most part on independent data sets and different sets of modeling assumptions. In order to evaluate such models meaningfully, methods are required to explicitly model the spatial certainty with which individual structural components are positioned by specific data sets. In this report, we use a constraint satisfaction algorithm to explicitly assess the location of the secondary structural elements of the 16S RNA, as well as the certainty with which these elements can be positioned. The algorithm initially assumes that these helical elements can occupy any position and orientation and then systematically eliminates those positions and orientations that do not satisfy formally parameterized interpretations of structural constraints. Using a conservative interpretation of the hydroxyl radical footprinting data, the positions of the ribosomal proteins as defined by neutron diffraction studies, and the secondary structure of 16S rRNA, the location of the RNA secondary structural elements can be defined with an average precision of 25 A (ranging from 12.8 to 56.3 A). The uncertainty in individual helix positions is both heterogeneous and dependent upon the number of constraints imposed on the helix. The topology of the resulting model is consistent with previous models based on independent approaches. The result of our computation is a conservative upper bound on the possible positions of the RNA secondary structural elements allowed by this data set, and provides a suitable starting point for refinement with other sources of data or different sets of modeling assumptions.

Base Sequence↗

PubMed1996

In vitro complementation analysis localizes 23S rRNA posttranscriptional modifications that are required for Escherichia coli 50S ribosomal subunit assembly and function.

In vitro transcripts of Escherichia coli 23S rRNA are compromised severely (at least five orders of magnitude below natural 23S rRNA) in their ability to reconstitute into catalytically active, correctly assembled 50S subunits in a standard reconstitution procedure. Denaturation experiments suggest that this deficiency is the result of missing posttranscriptional modifications present in natural 23S rRNA. An in vitro complementation analysis was performed where partial natural 23S rRNA fragments prepared by RNase H digestion or hammerhead ribozyme cleavage were combined with the remaining RNA as a partial in vitro transcript in a standard reconstitution reaction and the peptidyl transferase activity was measured. This approach has identified a ca. 80-nt region in 23S rRNA (extending from position 2445 to 2523) containing the natural RNA element essential for E. coli 50S subunit assembly and has excluded the requirement for all but six of the known posttranscriptional modifications in 23S rRNA for 50S subunit assembly or peptidyl transferase activity. Importantly, this chimeric reconstitution approach provides a system for the analysis of pure mutant populations of 23S rRNA reconstituted into 50S subunits.

Escherichia coli↗

PubMed1995

A base pair between tRNA and 23S rRNA in the peptidyl transferase centre of the ribosome.

Interaction of the conserved CCA terminus of tRNA with rRNA in the peptidyl transferase P site has been studied by in vitro genetics. A watson-Crick G-C pair between G2252 in a conserved hairpin loop of 23S rRNA and C74 at the acceptor end of tRNA is required for proper functional interaction of the CCA end of tRNA with the ribosomal P site. These findings establish a direct role for 23S rRNA in protein synthesis.

Base Composition↗

PubMed1995

Specific protection of 16 S rRNA by translational initiation factors.

The binding of initiation factors to 30 S ribosomal subunits protects specific sets of nucleotides in 16 S rRNA from base-specific chemical probes. Initiation factor 3 (IF-3) protects residues G700, G703 and G791 from attack by kethoxal. These protected bases are close to those in 16 S rRNA that are protected by 50 S subunits, providing a structural basis for the subunit dissociation activity of IF-3. The IF-3-dependent protections also flank bases that are protected by P-site-bound tRNA, in keeping with the possibility that IF-3 may interact with initiator tRNA, or influence the properties of the 30 S P site during initiation. IF-1 protects G530, A1492 and A1493 and causes enhanced reactivity of A1408. These bases are precisely the ones that are protected by the binding of tRNA to the ribosomal A site. This suggests that IF-1 mimics A-site-bound tRNA, and could serve to prevent premature binding of aminoacyl tRNA by blocking the 30 S A site. We were unable to detect any effect of IF-2 on the reactivity pattern of 16 S rRNA, suggesting that this factor may interact primarily through protein-protein interactions.

Aldehydes↗

PubMed1995

Suppression of a cold-sensitive mutation in 16S rRNA by overexpression of a novel ribosome-binding factor, RbfA.

A novel 15-kDa protein, RbfA, has been identified by virtue of its ability to act as a high copy suppressor of a previously characterized dominant cold-sensitive mutation (C23U) in 16S rRNA. RbfA is found associated with free 30S ribosomal subunits, but not with 70S ribosomes or polysomes, and is essential for maximal cell growth, particularly at low temperatures. Cells lacking RbfA in a wild-type rRNA background exhibit a cold-sensitive phenotype that is strikingly similar to that of the cold-sensitive C23U rRNA mutant. The observed patterns of allele specificity of suppression and synthetic lethality in cells containing an RbfA knockout in combination with various 16S rRNA mutations suggests that RbfA interacts with the 5'-terminal helix region of 16S rRNA, possibly during a late step of 30S maturation.

Amino Acid Sequence↗

PubMed1995

Directed hydroxyl radical probing of 16S rRNA using Fe(II) tethered to ribosomal protein S4.

Localized hydroxyl radical probing has been used to explore the rRNA neighborhood around a unique position in the structure of the Escherichia coli 30S ribosomal subunit. Fe(II) was attached to ribosomal protein S4 at Cys-31 via the reagent 1-(p-bromoacetamidobenzyl)-EDTA. [Fe-Cys31]S4 was then complexed with 16S rRNA or incorporated into active 30S ribosomal subunits by in vitro reconstitution with 16S rRNA and a mixture of the remaining 30S subunit proteins. Hydroxyl radicals generated from the tethered Fe resulted in cleavage of the 16S rRNA chain in two localized regions of its 5' domain. One region spans positions 419-432 and is close to the multihelix junction previously placed at the RNA binding site of S4 by chemical and enzymatic protection (footprinting) and crosslinking studies. A second site of directed cleavage includes nucleotides 297-303, which overlap a site that is protected from chemical modification by protein S16, a near neighbor of S4 in the ribosome. These results provide useful information about the three-dimensional organization of 16S rRNA and indicate that these two regions of its 5' domain are in close spatial proximity to Cys-31 of protein S4.

Escherichia coli↗

PubMed1995

A temperature-dependent conformational rearrangement in the ribosomal protein S4.16 S rRNA complex.

Ribosomal protein S4 protects a characteristic set of bases in 16 S rRNA from attack by chemical probes. Use of hydroxyl radical as a probe of the RNA backbone shows that ribose residues in these same regions are also protected by S4, confirming the localization of its interactions with 16 S rRNA to the junction of five helical elements in the proximal region of the 5' domain. At 0 degrees C, the nucleotides protected by S4 from base-specific probes are confined almost exclusively to the two compound helices formed by residues 404-499. After subsequent heating of the complex briefly at 30 or 42 degrees C, nucleotides in the three adjacent helices are additionally protected, resulting in a pattern of protection that is identical to that which is observed when S4 is incubated with 16 S rRNA under in vitro reconstitution conditions. Preincubation of the protein or the RNA (or both) separately at elevated temperature does not substitute for heating the S4.RNA complex. The regions in the RNA affected by the heat step are known to interact with proteins S12 and S16, both of which depend upon S4 for their binding to the RNA. Thus, the finding that S4 recruits additional sites of interaction in the RNA following its initial binding suggests a possible mechanism to insure the sequential addition of proteins during ribosomal assembly.

Cold Temperature↗

PubMed1995

Identification of bases in 16S rRNA essential for tRNA binding at the 30S ribosomal P site.

Previous studies suggest that the mechanism of action of the ribosome in translation involves crucial transfer RNA (tRNA)-ribosomal RNA (rRNA) interactions. Here, a selection scheme was developed to identify bases in 16S rRNA that are essential for tRNA binding to the P site of the small (30S) ribosomal subunit. Modification of the N-1 and N-2 positions of 2-methylguanine 966 and of the N-7 position of guanine 1401 interfered with messenger RNA (mRNA)-dependent binding of tRNA to the P site. Modification of the same positions as well as of the N-1 and N-2 positions of guanine 926 interfered with mRNA-independent binding of tRNA at high magnesium ion concentration. These results suggest that these three bases are involved in intermolecular contacts between ribosomes and tRNA.

Aldehydes↗

PubMed1995

Hydroxyl radical footprinting of ribosomal proteins on 16S rRNA.

Complexes between 16S rRNA and purified ribosomal proteins, either singly or in combination, were assembled in vitro and probed with hydroxyl radicals generated from free Fe(II)-EDTA. The broad specificity of hydroxyl radicals for attack at the ribose moiety in both single- and double-stranded contexts permitted probing of nearly all of the nucleotides in the 16S rRNA chain. Specific protection of localized regions of the RNA was observed in response to assembly of most of the ribosomal proteins. The locations of the protected regions were in good general agreement with the footprints previously reported for base-specific chemical probes, and with sites of RNA-protein crosslinking. New information was obtained about interaction of ribosomal proteins with 16S rRNA, especially with helical elements of the RNA. In some cases, 5' or 3' stagger in the protection pattern on complementary strands suggests interaction of proteins with the major or minor groove, respectively, of the RNA. These results reinforce and extend previous data on the localization of ribosomal proteins with respect to structural features of 16S rRNA, and offer many new constraints for three-dimensional modeling of the 30S ribosomal subunit.

Base Sequence↗

PubMed1994

Phylogenetic comparative chemical footprint analysis of the interaction between ribonuclease P RNA and tRNA.

Ribonuclease P RNA is the catalytic moiety of the ribonucleoprotein enzyme that endonucleolytically cleaves precursor sequences from the 5' ends of pre-tRNAs. The bacterial RNase P RNA-tRNA complex was examined with a footprinting approach, utilizing chemical modification to determine RNase P RNA nucleotides that potentially contact tRNA. RNase P RNA was modified with dimethylsulfate or kethoxal in the presence or absence of tRNA, and sites of modification were detected by primer extension. Comparison of the results reveals RNase P bases that are protected from modification upon binding tRNA. Analyses were carried out with RNase P RNAs from three different bacteria: Escherichia coli, Chromatium vinosum and Bacillus subtilis. Discrete bases of these RNAs that lie within conserved, homologous portions of the secondary structures are similarly protected. One protection among all three RNAs was attributed to the precursor segment of pre-tRNA. Experiments using pre-tRNAs containing precursor segments of variable length demonstrate that a precursor segment of only 2-4 nucleotides is sufficient to confer this protection. Deletion of the 3'-terminal CCA sequence of tRNA correlates with loss of protection of a particular loop in the RNase P RNA secondary structure. Analysis of mutant tRNAs containing sequential 3'-terminal deletions suggests a relative orientation of the bound tRNA CCA to that loop.

Bacillus subtilis↗

PubMed1994

Independent in vitro assembly of a ribonucleoprotein particle containing the 3' domain of 16S rRNA.

Small (30S) subunits of Escherichia coli ribosomes are composed of 21 proteins and a 1542-nucleotide 16S rRNA, whose secondary structure is divided into three domains. An in vitro transcript of the 3' domain of 16S rRNA (residues 923-1542), assembles efficiently with 30S ribosomal proteins to form a compact ribonucleoprotein (RNP) particle. Isolated particles examined under the electron microscope have a globular appearance, similar in size and shape to the head of the 30S ribosomal subunit. Two-dimensional gel analysis of the particles indicates the presence of proteins S3, S7, S9, S10, S13, S14, and S19 and smaller amounts of S2, all of which have been localized to the head of the 30S subunit by immunoelectron microscopy and neutron diffraction and belong to the S7 assembly family. Interestingly, protein S4, which is believed to interact exclusively with the 5' domain, is also reproducibly found associated with the particles in significant amounts. Chemical probing of the RNA in the assembled particle reveals characteristic cleavage protection patterns, showing that the proteins assemble with the 3'-domain RNA similarly to the way in which they assemble with 16S rRNA, although some of the later steps of assembly appear to be incomplete. These results show that the 3' domain of 16S rRNA can indeed assemble independently of the rest of the 30S subunit into a particle that resembles its structure in the ribosome. In addition, the assembled particles are able to bind spectinomycin with an affinity comparable to that of 30S subunits.

Base Sequence↗

PubMed1994

Footprinting mRNA-ribosome complexes with chemical probes.

We footprinted the interaction of model mRNAs with 30S ribosomal subunits in the presence or absence of tRNA(fMet) or tRNA(Phe) using chemical probes directed at the sugar-phosphate backbone or bases of the mRNAs. When bound to the 30S subunits in the presence of tRNA(fMet), the sugar-phosphate backbones of gene 32 mRNA and 022 mRNA are protected from hydroxyl radical attack within a region of about 54 nucleotides bounded by positions -35 (+/- 2) and +19, extending to position +22 when tRNA(Phe) is used. In 70S ribosomes, protection is extended in the 5' direction to about position -39 (+/- 2). In the absence of tRNA, the 30S subunit protects only nucleotides -35 (+/- 2) to +5. Introduction of a stable tetraloop hairpin between positions +10 and +11 of gene 32 mRNA does not interfere with tRNA(fMet)-dependent binding of the mRNA to 30S subunits, but results in loss of protection of the sugar-phosphate backbone of the mRNA downstream of position +5. Using base-specific probes, we find that the Shine-Dalgarno sequence (A-12, A-11, G-10 and G-9) and the initiation codon (A+1, U+2 and G+3) of gene 32 mRNA are strongly protected by 30S subunits in the presence of initiator tRNA. In the presence of tRNA(Phe), the same Shine-Dalgarno bases are protected, as are U+4, U+5 and U+6 of the phenylalanine codon. Interestingly, A-1, immediately preceding the initiation codon, is protected in the complex with 30S subunits and initiator tRNA, while U+2 and G+3 are protected in the complex with tRNA(Phe) in the absence of initiator tRNA. Additionally, specific bases upstream from the Shine-Dalgarno region (U-33, G-32 and U-22) as well as 3' to the initiation codon (G+11) are protected by 30S subunits in the presence of either tRNA. These results imply that the mRNA binding site of the 30S subunit covers about 54-57 nucleotides and are consistent with the possibility that the ribosome interacts with mRNA along its sugar-phosphate backbone.

Bacteriophage T7↗

PubMed1994

A conserved secondary structural motif in 23S rRNA defines the site of interaction of amicetin, a universal inhibitor of peptide bond formation.

The binding site and probable site of action have been determined for the universal antibiotic amicetin which inhibits peptide bond formation. Evidence from in vivo mutants, site-directed mutations and chemical footprinting all implicate a highly conserved motif in the secondary structure of the 23S-like rRNA close to the central circle of domain V. We infer that this motif lies at, or close to, the catalytic site in the peptidyl transfer centre. The binding site of amicetin is the first of a group of functionally related hexose-cytosine inhibitors to be localized on the ribosome.

Anti-Bacterial Agents↗