PubMed Health⌕ Search

Biomedical subjects

B Hardesty

Publications and source records attributed to B Hardesty.

At least 19 recordsLinked to original sources
PubMed1992

Evidence for RNA in the peptidyl transferase center of Escherichia coli ribosomes as indicated by fluorescence.

A coumarin derivative was covalently attached to either the amino acid or the 5' end of phenylalanine-specific transfer RNA (tRNA(phe)). Its fluorescence was quenched by methyl viologen when the tRNA was free in solution or bound to Escherichia coli ribosomes. Methyl viologen as a cation in solution has a strong affinity for the ionized phosphates of a nucleic acid and so can be used to qualitatively measure the presence of RNA in the immediate vicinity of the tRNA-linked coumarins upon binding to ribosomes. Fluorescence lifetime measurements indicate that the increase in fluorescence quenching observed when the tRNAs are bound into the peptidyl site of ribosomes is due to static quenching by methyl viologen bound to RNA in the immediate vicinity of the fluorophore. The data lead to the conclusion that the ribosome peptidyl transferase center is rich in ribosomal RNA. Movement of the fluorophore at the N-terminus of the nascent peptide as it is extended or movement of the tRNA acceptor stem away from the peptidyl transferase center during peptide bond formation appears to result in movement of the probe into a region containing less rRNA.

Escherichia coli↗
PubMed1992

High efficiency cell-free synthesis of proteins: refinement of the coupled transcription/translation system.

Two modifications are introduced to convert the Escherichia coli cell-free extract ("S30") into a high efficiency system for coupled transcription/translation of exogenously added genes. (a) The ribosome fraction collected from the S30 by ultracentrifugation is used. It contains all the proteins necessary for gene expression but has lost the vast majority of soluble proteins that might interfere with purification and enzymatic activity of product formed. (b) Plasmids containing coding sequences to be expressed are not linearized thus enhancing their stability by avoiding their degradation. These two modifications not only improve protein synthesis in a static system but allow gene expression over 20-40 h in the continuous-flow cell-free system. Both prokaryotic and eukaryotic proteins have been synthesized in this system.

Carbon Radioisotopes↗
PubMed1992

Use of 50 S-binding antibiotics to characterize the ribosomal site to which peptidyl-tRNA is bound.

Five antibiotics (puromycin, erythromycin, lincomycin, sparsomycin, and virginiamycin M1) that bind specifically to the 50 S ribosomal subunit near the peptidyl transferase center were used to compare and characterize the positions of bound AcylPhe-tRNA in the puromycin-reactive and -unreactive states. Binding of the antibiotics was quantitatively measured by their perturbation of fluorescence from probes attached to the alpha-amino group of Phe-tRNA. Derivatives of three probes with differing chemical characteristics and environmental sensitivities were used: a coumarin, an aminonaphthalenesulfonate, and a pyrene. The effects of the antibiotics on the fluorescence of labeled AcylPhe-tRNAs in the two states, while generally qualitatively similar, are nonetheless quantitatively distinct, as are the calculated binding constants for the antibiotics. Puromycin, as reported earlier, binds to both the puromycin-reactive and -unreactive states, but its dissociation constant is higher for the latter state. Erythromycin binds tightly to ribosomes bearing labeled AcylPhe-tRNA in either the puromycin-reactive or -unreactive state. Its effect on the fluorescence of the labeled tRNA is very similar in the two states, except with the pyrene probe, where it has a larger effect in the puromycin-reactive state. Lincomycin and sparsomycin bind to both ribosomal states, but both bind more tightly to the puromycin-reactive state, the extent of the difference varying with the identity of the fluorescent probe. Virginiamycin M1 binds to ribosomes with AcylPhe-tRNA in the puromycin-reactive site, but its binding could not be detected to ribosomes with AcylPhe-tRNA in the puromycin-unreactive site.

Anti-Bacterial Agents↗
PubMed1992

Fluorescence characterization of the environment encountered by nascent polyalanine and polyserine as they exit Escherichia coli ribosomes during translation.

The fate of the amino termini of nascent polyalanine, polyserine, and polylysine was monitored by fluorescence techniques as each was translated on Escherichia coli ribosomes. A coumarin probe was placed at the alpha-amino group of a synthetic elongator alanyl-tRNA or a synthetic initiator alanyl-tRNA or at the epsilon-amino group of natural lysyl-tRNA, and each was used to nonenzymatically initiate peptide synthesis. The fluorescent alanyl-tRNAs containing an AAA anticodon were used to initiate polyserine (with a synthetic tRNA(Ser] or polyalanine synthesis from a poly(uridylic acid) template. The fluorescent lysyl-tRNA was used to initiate polylysine synthesis from poly(adenylic acid). Changes in the fluorescence of the amino-terminal coumarin were examined to characterize the environment of the probe as the nascent peptides were extended. Protection from proteolysis and the binding of anti-coumarin antibodies or Fab fragments suggest that the amino terminus of each polypeptide is protected from interaction with proteins (Mr greater than 28,000) until the peptides are extended to an average length of 40-50 residues; however, the fluorescence from the amino terminus of shorter nascent polyalanine and polyserine peptides was readily quenched by methyl viologen (Mr = 257), indicating ribosomes do not shield the nascent peptide from molecules of this size. The data appear to indicate that polyalanine, polyserine, and polylysine are extended from the peptidyl transferase into a protected region of the ribosome such as a groove or tunnel but that this region is readily accessible to small molecules.

Amino Acid Sequence↗
PubMed1992

Ribosome function determined by fluorescence.

Five different fluorescence phenomena are considered in relation to their use to study the structure and function of ribosomes. These are: quantum yield or emission intensity; emission wavelength maximum; fluorescence anisotropy; collisional quenching; and nonradiative energy transfer. Results from a number of studies in which these techniques were used are described and summarized in relation to the movement and conformation of tRNA, the nascent peptide, and mRNA in a ribosome during the reaction steps of peptide elongation.

Base Sequence↗
PubMed1991

A synthetic alanyl-initiator tRNA with initiator tRNA properties as determined by fluorescence measurements: comparison to a synthetic alanyl-elongator tRNA.

Two synthetic tRNAs have been generated that can be enzymatically aminoacylated with alanine and have AAA anticodons to recognize a poly(U) template. One of the tRNAs (tRNA(eAla/AAA)) is nearly identical to Escherichia coli elongator tRNA(Ala). The other has a sequence similar to Escherichia coli initiator tRNA(Met) (tRNA(iAla/AAA)). Although both tRNAs can be used in poly(U)-directed nonenzymatic initiation at 15 mM Mg2+, only the elongator tRNA can serve for peptide elongation and polyalanine synthesis. Only the initiator tRNA can be bound to 30S ribosomal subunits or 70S ribosomes in the presence of initiation factor 2 (IF-2) and low Mg2+ suggesting that it can function in enzymatic peptide initiation. A derivative of coumarin was covalently attached to the alpha amino group of alanine of these two Ala-tRNA species. The fluorescence spectra, quantum yield and anisotropy for the two Ala-tRNA derivatives are different when they are bound to 70S ribosomes (nonenzymatically in the presence of 15 mM Mg2+) indicating that the local environment of the probe is different. Also, the effect of erythromycin on their fluorescence is quite different, suggesting that the probes and presumably the alanine moiety to which they are covalently linked are in different positions on the ribosomes.

Base Sequence↗
PubMed1991

Fluorescence studies on the interaction of inhibitor 2 and okadaic acid with the catalytic subunit of type 1 phosphoprotein phosphatases.

Phosphatase inhibitor 2 was mutagenized and expressed in Escherichia coli to produce a protein with a single cysteinyl residue at position 129. The newly introduced sulfhydryl group was labeled with a maleimide derivative of coumarin (CPM). The resulting fluorescent inhibitor 2 molecule (CPM-I2) retains biological activity and binds to the catalytic subunit of type 1 phosphatase (PP1-C) with a Kd similar to the Ki of native I2 (2-3 nM). Fluorescence anisotropy data indicate that kinase FA (glycogen synthase kinase 3) does not dissociate the CPM-I2.PP1-C complex but rather causes a conformational change in the I2 molecule that is retained even after the CPM-I2 is displaced by an excess of native I2. The fluorescence data presented here also indicate that okadaic acid and I2 are competitive for binding to PP1-C, even after kinase FA treatment of the CPM-I2.PP1-C complex.

Adenosine Triphosphate↗
PubMed1991

The synthesis of polyphenylalanine on ribosomes to which erythromycin is bound.

Erythromycin binds to the large subunit of Escherichia coli ribosomes at a specific site that is very close to the amino acid of aminoacyl-tRNA bound into the peptidyltransferase center, and to the site to which puromycin is bound, the P and A sites, respectively, of the classical two-site model of ribosome function. Both erythromycin and puromycin affect fluorescence from fluorescent derivatives of aminoacyl-tRNAs, while both puromycin and aminoacyl-tRNAs affect fluorescence of fluorescent derivatives of erythromycylamine. The results demonstrate unequivocally that erythromycin, deacylated tRNA, a peptidyl-tRNA analogue and puromycin can be bound simultaneously to the same ribosome. Nascent peptides of more than a few amino acids in length block binding of erythromycin to the ribosomes but, unlike most other peptides, long polyphenylalanine chains can be synthesized on ribosomes to which erythromycin is bound. It is suggested that this refractory synthesis in the presence of erythromycin reflects the atypical physical and structural properties of polyphenylalanine.

Binding Sites↗
PubMed1991

Fluorescence study of the topology of messenger RNA bound to the 30S ribosomal subunit of Escherichia coli.

Short RNAs (25-36 nucleotides in length) with sequences of the translational initiation region of bacteriophage R17 protein A mRNA were produced by chemical and in vitro transcription techniques and labeled at their 5' or 3' ends with fluorescent probes. The interaction of these labeled RNAs with the 30S subunit of Escherichia coli was studied by using fluorescence spectroscopic techniques. All the RNAs bound tightly to 30S subunits (Kd less than or equal to 200 nM). Resonance energy transfer experiments demonstrated the proximity of the ends of the RNAs to each other and to two fluorescently labeled sites on the 30S subunit: the 3' end of 16S rRNA and the cysteine residue of ribosomal protein S21. By using the distances calculated from energy transfer between the 3' end of 16S rRNA and the ends of RNAs of varying lengths, a topological map of this region of mRNA on the 30S subunit was constructed.

Base Sequence↗
PubMed1991

Eukaryotic protein synthesis initiation factor 2. A target for inactivation by proanthocyanidin.

Polyproanthocyanidin (PPA), a phenolic polymer isolated from the plant Alhagi kirgisorum S. was found to interact strongly with eukaryotic initiation factor 2 (eIF-2), thereby inhibiting reactions involving this protein. When added to a rabbit reticulocyte lysate system, PPA blocks in vitro translation and it appears to selectively bind and precipitate a relatively small number of proteins including eIF-2 and regulin. The phosphorylation of purified regulin and eIF-2 by casein kinase II (CK II) and the heme-sensitive eIF-2 alpha kinase, respectively, was also inhibited by the polyphenolic compound. The natural fluorescence of PPA was utilized to compare its interaction with eIF-2 and regulin to that with other natural and synthetic polypeptides.

Animals↗
PubMed1991

The conformation of nascent polylysine and polyphenylalanine peptides on ribosomes.

Polypeptide synthesis using either phenylalanine or lysine was initiated on Escherichia coli ribosomes; then the position and conformation of the nascent peptide were monitored by fluorescence techniques. To this end, fluorophores had been attached to the amino terminus of each nascent peptide, and major differences were observed as chain extension occurred. Polyphenylalanine appeared to build up as a hydrophobic mass adjacent to the peptidyl transferase center while polylysine apparently was extended directly from the ribosome into the surrounding solution. An explanation for these differences may be provided by the physical and chemical properties of each polypeptide. These properties may be responsible for the route by which each peptide exits the peptidyl transferase center as demonstrated by the different sensitivity of each to inhibition by erythromycin.

Dipeptides↗
PubMed1990

Movement of tRNA but not the nascent peptide during peptide bond formation on ribosomes.

The results from experiments involving nonradiative energy transfer indicate that a fluorescent probe on the 5'-end of tRNA(Phe) moves more than 20 A towards probes on ribosomal protein L1 as a peptide bond is formed during the peptidyl transferase reaction on Escherichia coli ribosomes. The peptide itself moves no more than a few angstroms during peptide bond formation, as judged by the movement of fluorescent probes attached to the phenylalanine amino group of phenylalanyl-tRNA. Other results demonstrate that an analogue of peptidyl-tRNA, deacylated tRNA, and puromycin can be bound simultaneously to the same ribosome, indicating that there are three physically distinct sites to which tRNA is bound during the reaction steps by which peptides are elongated. The results appear to be consistent with the displacement model of peptide elongation.

Bacterial Proteins↗
PubMed1990

Three phosphorylation sites in elongation factor 2.

Elongation factor 2 (EF-2) of rabbit reticulocytes was phosphorylated in vitro by incubation with partially purified EF-2 kinase and [gamma-32P]ATP. After exhaustive tryptic hydrolysis 4 phosphopeptides were revealed by two-dimensional peptide mapping. The phosphopeptides were isolated by high performance liquid chromatography and sequenced. A comparison of the primary structure of the phosphopeptides with that of EF-2 showed that all 4 phosphopeptides originated from one region of EF-2 located near the N-terminus that contains 3 threonine residues: Thr-53, Thr-56, Thr-58. A direct estimation of localization of radioactive phosphate in the phosphopeptides demonstrated that all the enumerated threonine residues in EF-2 can be phosphorylated in vitro.

Adenosine Triphosphate↗
PubMed1990

The extension of polyphenylalanine and polylysine peptides on Escherichia coli ribosomes.

Fluorescence techniques were used to examine aminoacyl-tRNA binding to Escherichia coli ribosomes and the subsequent extension of polyphenylalanine and polylysine nascent peptides. The results demonstrate that deacylated tRNA, an analogue of peptidyl-tRNA and puromycin (an analogue of aminoacyl-tRNA) can be bound simultaneously to the same ribosome. Moreover, the fluorescence properties of nascent polyphenylalanine and polylysine peptides with a fluorophore attached to their amino termini were determined and found to be quite different. This difference is reflected in the effects that erythromycin has in each case.

Binding Sites↗
PubMed1979

No effect of cAMP on protein synthesis in reticulocyte lysates.

The results of a series of experiments are interpreted to indicate that protein synthesis in reticulocyte lysates is not affected by the reticulocyte cAMP-dependent protein kinase. The catalytic subunit of this enzyme was isolated to apparent homogeneity. Also, the protein inhibitor of this protein kinase was isolated from muscle. Neither physiological concentrations of cAMP nor any of these protein components had a detectable effect on protein synthesis in reticulocyte lysates in the presence or absence of exogenous heme. Phosphorylation of the smallest subunit of eukaryotic initiation factor 2 or the 90,000 to 100,000-dalton peptide associated with eukaryotic initiation factor 2 kinase activity were not affected by the activity of the cAMP-dependent protein kinase under conditions in which exogenous heme has a pronounced effect on these reactions.

Animals↗