Biomedical subjects

B Hardesty

Publications and source records attributed to B Hardesty.

At least 37 records · Page 2Linked to original sources

PubMed1997

Ribosomes and ribosomal RNA as chaperones for folding of proteins.

BACKGROUND: Provocative recent reports indicate that the large subunits of either prokaryotic or eukaryotic ribosomes have the capacity to promote refolding of denatured enzymes. RESULTS: Salt-washed Escherichia coli ribosomes are shown to promote refolding of denatured rhodanese. The ability of the ribosomes to carry out renaturation is a property of the 50S ribosomal subunit, specifically the 23S rRNA. Refolding and release of enzymatically active rhodanese leaves the ribosomes in an inactive state or conformation for subsequent rounds refolding. Inactive ribosomes can be activated by elongation factor G (EF-G) plus GTP or by cleavage of their 23S rRNA by alpha-sarcin. Activation by either mechanism is strongly inhibited by the EF-G.GDP.fusidic acid complex. CONCLUSIONS: Large subunits of E. coli ribosomes, specifically 23S rRNA, have the capacity to mediate refolding of denatured rhodanese. Refolding activity is related to the state or conformation of ribosomes that is promoted by EF-G. Activation by either mechanism is strongly inhibited by the EF-G.GDP.fusidic acid complex.

Endoribonucleases↗

PubMed1996

Binding of an N-terminal rhodanese peptide to DnaJ and to ribosomes.

A peptide corresponding to the N-terminal 17 amino acids of bovine rhodanese was fluorescently labeled with a coumarin derivative at its primary amino group(s) and then purified by high performance liquid chromatography. This peptide interacted with the molecular chaperone DnaJ in the absence of other chaperones and ATP. In the presence of ATP, the molecular chaperone DnaK bound to the DnaJ-peptide complex, but not to the peptide alone. The chaperone GrpE appeared to cause the release of the peptide bound to the ternary complex in the presence of ATP but not in the presence of ADP. This nucleotide apparently stabilized the complex. The peptide also bound to salt-washed Escherichia coli 70 S ribosomes, specifically to 50 S ribosomal subunits, not to 30 S subunits. DnaJ plus DnaK interacted with the peptide on the ribosome. GrpE caused dissociation of the peptide from the ribosome; ATP was required for this reaction. It was inhibited by ADP. A comparable series of chaperone-mediated reactions is assumed to occur with the N-terminal segment of the nascent polypeptide to facilitate its folding on ribosomes.

Adenosine Diphosphate↗

PubMed1995

Folding of an enzyme into an active conformation while bound as peptidyl-tRNA to the ribosome.

Rhodanese bound to bacterial ribosomes as peptidyl-tRNA can be folded into an enzymatically active conformation by generating C-terminal extensions of the wild-type enzyme. Rhodanese was synthesized by coupled transcription/translation in a cell-free Escherichia coli system from plasmids containing the coding sequences for the wild-type enzyme or its C-terminally extended mutants. Two proteins with extensions of 23 amino acids or longer were enzymatically active while bound to the ribosomes whereas wild-type protein and a 13-amino acid extension were not. All forms of the enzyme were active after termination and release of the full-length protein from the ribosomes. All five of the bacterial chaperones were required to substantially increase the specific enzymatic activity of the extended rhodanese while the nascent protein was bound to ribosomes. The results provide direct support for the hypothesis that proteins acquire tertiary structure as they are formed in ribosomes.

Cell-Free System↗

PubMed1995

Elongation and folding of nascent ricin chains as peptidyl-tRNA on ribosomes: the effect of amino acid deletions on these processes.

Ricin A-chain was used as a test protein to study the effects of deletion of codons on the ribosomal synthesis, release and chaperone-mediated folding of the proteins. Synthesis of wild-type ricin and five mutant proteins was carried out in an Escherichia coli cell-free coupled transcription/translation system from otherwise identical non-linearized plasmids. The deletions involved small numbers of contiguous amino acid residues at different points from the N terminus to the C terminus of the wild-type protein. Deletion of the N-terminal 20 amino acid residues caused a 45% reduction in total protein synthesis whereas deletion of the next three amino acid residues caused a 1.5-fold increase in synthesis compared with wild-type with an accumulation of full-length polypeptides as peptidyl-tRNA in the ribosomal P site. Intermediate levels of synthesis and release were seen with the other three mutants. Enzymatic activity was detected only with wild-type protein and a mutant lacking the C-terminal five amino acid residues. These were the only ricin species in which chaperone-dependent reactions could be detected by fluorescence from coumarin incorporated with methionine at the N terminus of the proteins. By using sparsomycin to block termination of full-length peptidyl-tRNA, it was demonstrated that the chaperone-mediated reactions detected by fluorescence occur on the ribosomes and involve folding of the nascent protein as peptidyl-tRNA. The results presented provide a direct demonstration of two points of fundamental importance: folding of nascent proteins involving chaperone-mediated reactions can occur on ribosomes and is directly related to the conformation of the native enzyme. Deletion of amino acid residues at different points from the N terminus to the C terminus affects the reactions of elongation, chaperone-mediated folding and release of full-length protein.

Cell-Free System↗

PubMed1995

The importance of the N-terminal segment for DnaJ-mediated folding of rhodanese while bound to ribosomes as peptidyl-tRNA.

Two lines of evidence indicate the importance of the N-terminal portion of rhodanese for correct folding of the nascent ribosome-bound polypeptide. A mutant gene lacking the codons for amino acids 1-23 of the wild-type protein is expressed very efficiently by coupled transcription/translation on Escherichia coli ribosomes; however, the mutant protein that is released from the ribosomes is enzymatically inactive. The mutant protein does not undergo the reaction that is promoted by the bacterial chaperone, DnaJ, which appears to be essential for folding of ribosome-bound rhodanese into the native conformation. The effect of DnaJ is monitored by fluorescence from coumarin cotranslationally incorporated at the N terminus of nascent rhodanese. Secondly, a synthetic peptide corresponding to the N-terminal 17 amino acids of the wild-type protein interferes with the synthesis of wild-type rhodanese but has much less effect on the synthesis of the N-terminal deletion mutant. The N-terminal peptide inhibits the effect of DnaJ on the nascent wild-type rhodanese and blocks the chaperone-mediated release and activation of ribosome-bound full-length rhodanese polypeptides that accumulate during in vitro synthesis. The results lead to the hypothesis that the N-terminal segment of rhodanese is required for its chaperone-dependent folding on the ribosome.

Escherichia coli Proteins↗

PubMed1995

Inhibition of the release factor-dependent termination reaction on ribosomes by DnaJ and the N-terminal peptide of rhodanese.

A peptide consisting of the 17 N-terminal amino acids of native bovine rhodanese in combination with the chaperone DnaJ specifically inhibits release factor- and stop codon-dependent hydrolysis of N-formylmethionine from N(formyl)-methionyl-tRNA bound with AUG to salt-washed ribosomes. Neither the peptide nor DnaJ by itself causes this inhibition. The N-terminal peptide and DnaJ both singularly and combined do not affect the peptidyltransferase reaction per se. The total amount of rhodanese synthesized in the cell-free coupled transcription-translation system is reduced by the peptide, with concomitant accumulation of full-length enzymatically inactive rhodanese polypeptides on ribosomes. In combination with DnaJ, the N-terminal polypeptide inhibits the termination and release of full-length rhodanese peptides that have accumulated on Escherichia coli ribosomes during the course of uninhibited coupled transcription-translation in the cell-free system. This inhibition appears to involve release factor 2-mediated termination at the UGA termination codon in the coding sequence for rhodanese. It is suggested that the N-terminal peptide inhibits the binding of the release factor to ribosomes. These data appear to provide the first report of differential inhibition of the termination reaction on ribosomes without inhibition of the peptidyltransferase reaction and peptide elongation.

Amino Acid Sequence↗

PubMed1994

Chaperone-dependent folding and activation of ribosome-bound nascent rhodanese. Analysis by fluorescence.

Fluorescently labeled rhodanese was synthesized by coupled transcription/translation in a cell-free Escherichia coli system. A derivative of coumarin was co-translationally incorporated at the N terminus of the polypeptide. Molecules released from the ribosomes during the incubation are enzymatically active; however, continued incubation results in accumulation of enzymatically inactive full-length rhodanese polypeptides on the ribosomes. These can be activated and released in the presence of the added chaperones, DnaJ, DnaK, GrpE, GroEL, GroES and ATP. Fluorescence parameters (quantum yield, anisotropy and the emission maximum) of ribosome-bound coumarin-labeled rhodanese are affected differentially by addition of the chaperones individually or sequentially. Rhodanese released from the ribosomes in the presence of all chaperones (enzymatically active) differs in fluorescence properties from rhodanese released by GroES or DnaK only or by puromycin (enzymatically inactive) indicating a difference in conformation. Using sparsomycin, an inhibitor of the peptidyl transferase reaction, full-length rhodanese can be trapped on the ribosomes. A ribosome-bound intermediate formed by DnaJ or DnaJ plus DnaK was demonstrated by the effect of these chaperones on fluorescence spectra resulting from binding of anticoumarin antibodies to the N terminus of newly synthesized rhodanese. The results support the hypothesis that folding of nascent proteins can take place on the ribosome.

Chaperonins↗

PubMed1994

Activation and release of enzymatically inactive, full-length rhodanese that is bound to ribosomes as peptidyl-tRNA.

Synthesis of rhodanese in a cell-free coupled transcription/translation system derived from Escherichia coli leads to an accumulation of full-length rhodanese protein on the ribosomes as well as to enzymatically active protein that is released from the ribosomes into the supernatant fraction. The ribosome-bound protein is enzymatically inactive but can be activated and released from the ribosomes without additional protein synthesis by subsequent incubation in the presence of the added chaperones DnaJ, DnaK, GrpE, GroEL, and GroES plus ATP. Efficient activation requires that all of the chaperones are present together during incubation which yields fully active rhodanese. Incubation in the presence of DnaJ only inhibits release whereas incubation with only GroES or DnaK promotes the release of enzymatically inactive protein. Incubation of the ribosome with puromycin leads to the release of enzymatically inactive protein whereas release and activation in the presence of all of the chaperones is blocked by sparsomycin. The effect of these antibiotics provides very strong evidence that enzymatically inactive, full-length rhodanese is bound to the ribosomes as peptidyl-tRNA and that the peptidyl transferase reaction is required for its release. Considered together, the data indicate that chaperone-mediated late stages of rhodanese folding into the enzymatically active, native conformation are intimately associated with the process of termination and release that occurs as part of the reaction cycle of protein synthesis.

Animals↗

PubMed1994

Development of a chaperone-deficient system by fractionation of a prokaryotic coupled transcription/translation system.

A coupled transcription/translation system from Escherichia coli has been developed that is very active for protein synthesis but deficient in chaperone proteins. The chaperones GroEL and DnaK distribute during the first ultracentrifugation of the E. coli extract partially with the ribosomes and partially in a liquid, viscous fraction above the ribosomes. Gel filtration chromatography of this latter fraction separates GroEL and DnaK as high-molecular-weight components from the peak of activity of the factors required for protein synthesis. Thus, a chaperone-deficient transcription/translation system can be reconstituted with salt-washed ribosomes. This chaperone-deficient system was used to study synthesis and folding of bacterial dihydrofolate reductase and of rhodanese, a eukaryotic mitochondrial enzyme. Both enzymes were synthesized from nonlinearized plasmids that had the respective coding sequence under the SP6 promoter. Both enzymes were synthesized in active form and with high specific activity in the chaperone-deficient system. A high proportion, about 20% of newly synthesized dihydrofolate reductase and about 50% of rhodanese, stayed with the ribosomes after coupled transcription/translation. No enzymatic activity was detected in this fraction. Addition of the chaperones GroEL/ES and DnaK resulted in a shift of rhodanese molecules from the ribosomes into the supernatant fraction. Nearly all molecules in the supernatant were enzymatically active.

Bacterial Proteins↗

PubMed1993

In vitro protein engineering using synthetic tRNA(Ala) with different anticodons.

The use of synthetic tRNA for in vitro protein engineering was tested in a coupled transcription/translation system prepared from Escherichia coli. DNA sequences similar to the natural tRNA(Ala/UGC) gene from E. coli but with different anticodons were synthesized in vitro, cloned into a DNA plasmid, and then transcribed in vitro with T7 RNA polymerase. The UGC alanine anticodon was changed to CUA corresponding to the UAG stop codon, CCU corresponding to the rarely used AGG arginine codon, and two four-nucleotide anticodons used to suppress stop codons. Bacterial dihydrofolate reductase was the test protein. Its cloned coding sequence was mutagenized at the GUG codon for valine-75 to correspond to the anticodons of the tRNA constructs, and then the plasmids were used to direct the synthesis of dihydrofolate reductase in the coupled transcription/translation system containing the corresponding synthetic tRNA. The results indicate that all four synthetic tRNAs were functionally active in the synthesis of full-length, enzymatically active dihydrofolate reductase protein.

Amino Acid Sequence↗

PubMed1993

GroEL and GroES increase the specific enzymatic activity of newly-synthesized rhodanese if present during in vitro transcription/translation.

Enzymatically active mammalian rhodanese, a mitochondrial matrix enzyme, which has been found to require assistants for efficient refolding in vitro, has been synthesized from a plasmid in a cell-free, fractionated, coupled transcription/translation system derived from Escherichia coli. The bacterial chaperonins, GroEL and GroES, along with the rhodanese substrate thiosulfate greatly enhance the specific enzymatic activity of the rhodanese polypeptide that is formed. Indirect evidence suggests that the effect of the GroEL/ES chaperonins is on ribosome-bound nascent peptides. The in vitro transcription/translation system produces sufficient amounts of rhodanese to provide a system for studying factors that control the initial steps in folding of nascent proteins.

Bacterial Proteins↗

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↗