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1.
《Gene》1996,179(1):157-162
The chloramphenicol (Cm)-inducible cat and cmlA genes are regulated by translation attenuation, a regulatory device that modulates mRNA translation. In this form of gene regulation, translation of the CmR coding sequence is prevented by mRNA secondary structure that sequesters its ribosome-binding site (RBS). A translated leader of nine codons precedes the secondary structure, and induction results when a ribosome becomes stalled at a specific site in the leader. Here we demonstrate that the site of ribosome stalling in the leader is selected by a cis effect of the nascent leader peptide on its translating ribosome. 相似文献
2.
Harold Goff 《Biochimica et Biophysica Acta (BBA)/General Subjects》1978,542(2):348-355
13C nuclear magnetic resonance spectra have been obtained for variety of high-spin iron(III) porphyrin compounds and corresponding μ-oxo-bridged dimeric species. Large hyperfine shifts and significant line broadening are observed. The monomeric exhibit hyperfine shifts which are downfield with te exception of an upfield shift for the meso-carbon atom. Possible unpaired spin delocalization mechanisms and prospects for observing 13C NMR porphyrin resonances in high-spin ferrihemoproteins are discussed. Spectra reported here provide strategy for incorporation of 13C labels in hemoproteins either by biosynthetic or chemical means. The vinyl-CH2 resonances of iron(III) protoporphyrin IX located 260 parts per million downfield from tetramethylsilane are especially attractive from the standpoint of chemical labeling. 相似文献
3.
A conserved chloramphenicol binding site at the entrance to the ribosomal peptide exit tunnel 总被引:1,自引:0,他引:1
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The antibiotic chloramphenicol produces modifications in 23S rRNA when bound to ribosomes from the bacterium Escherichia coli and the archaeon Halobacterium halobium and irradiated with 365 nm light. The modifications map to nucleotides m5U747 and C2611/C2612, in domains II and V, respectively, of E.coli 23S rRNA and G2084 (2058 in E.coli numbering) in domain V of H.halobium 23S rRNA. The modification sites overlap with a portion of the macrolide binding site and cluster at the entrance to the peptide exit tunnel. The data correlate with the recently reported chloramphenicol binding site on an archaeal ribosome and suggest that a similar binding site is present on the E.coli ribosome. 相似文献
4.
5.
Phosphorus NMR spectra of natural DNA fragments in the course of the B-to-A conformational transition 总被引:1,自引:0,他引:1
Changes in the 31P-nmr spectra of sonicated natural DNA fragments were investigated in ethanol solutions where the fragments underwent, as checked by CD, the B-to-A conformational transition. The study produced the following conclusions: (1) The high DNA concentrations used for the 31P-nmr measurements promote the transition compared to dilute solutions that are commonly used for CD measurements. (2) The B-to-A transition was reflected in a cooperative downfield shift of the DNA 31P-nmr resonance, consistent with unwinding of the double helix. (3) Prior to the transition, the changes in chemical shift of double-and single-stranded DNAs were almost identical. It thus appears that the effect of ethanol on the geometry and hydration of phosphodiester linkages does not depend heavily on DNA base–base interactions. (4) The A-form resonances were 30–40% narrower than the B-form resonances, which is attributed to marked sequence-dependent variations in the latter conformation and to their reduction in the former. (5) The B-form DNA aggregated in the concentrated 31P-nmr samples in the presence of ethanol, judged from a milky opalescence of the solution and a substantial broadening of its 31P-nmr resonance. The broadening abruptly disappeared as soon as DNA adopted the A-form so that DNA, in dependence on the secondary structure, showed different tendencies to condense in the presence of ethanol. The condensation increased cooperativity of the B-to-A interconversion. 相似文献
6.
Gabriel J. FuenteGmez Creighton L. Kellum Alexis C. Miranda Michael R. Duff Jr Elizabeth E. Howell 《Protein science : a publication of the Protein Society》2021,30(2):477
R67 dihydrofolate reductase (R67 DHFR) is a plasmid‐encoded enzyme that confers resistance to the antibacterial drug trimethoprim. R67 DHFR is a tetramer with a single active site that is unusual as both cofactor and substrate are recognized by symmetry‐related residues. Such promiscuity has limited our previous efforts to differentiate ligand binding by NMR. To address this problem, we incorporated fluorine at positions 4, 5, 6, or 7 of the indole rings of tryptophans 38 and 45 and characterized the spectra to determine which probe was optimal for studying ligand binding. Two resonances were observed for all apo proteins. Unexpectedly, the W45 resonance appeared broad, and truncation of the disordered N‐termini resulted in the appearance of one sharp W45 resonance. These results are consistent with interaction of the N‐terminus with W45. Binding of the cofactor broadened W38 for all fluorine probes, whereas substrate, dihydrofolate, binding resulted in the appearance of three new resonances for 4‐ and 5‐fluoroindole labeled protein and severe line broadening for 6‐ and 7‐fluoroindole R67 DHFR. W45 became slightly broader upon ligand binding. With only two peaks in the 19F NMR spectra, our data were able to differentiate cofactor and substrate binding to the single, symmetric active site of R67 DHFR and yield binding affinities. 相似文献
7.
Shang-Te Danny Hsu Lisa D. Cabrita Paola Fucini Christopher M. Dobson 《Journal of molecular biology》2009,388(4):865-108
We have carried out a detailed structural and dynamical characterisation of the isolated fifth repeat of the gelation factor (ABP-120) from Dictyostelium discoideum (ddFLN5) by NMR spectroscopy to provide a basis for studies of co-translational folding on the ribosome of this immunoglobulin-like domain. The isolated ddFLN5 can fold autonomously in solution into a structure that resembles very closely the crystal structure of the domain in a construct in which the adjacent sixth repeat (ddFLN6) is covalently linked to its C-terminus in tandem but deviates locally from a second crystal structure in which ddFLN5 is flanked by ddFLN4 and ddFLN6 at both N- and C-termini. Conformational fluctuations were observed via 15N relaxation methods and are primarily localised in the interstrand loops that encompass the C-terminal hemisphere. These fluctuations are distinct in location from the region where line broadening is observed in ddFLN5 when attached to the ribosome as part of a nascent chain. This observation supports the conclusion that the broadening is associated with interactions with the ribosome surface [Hsu, S. T. D., Fucini, P., Cabrita, L. D., Launay, H., Dobson, C. M. & Christodoulou, J. (2007). Structure and dynamics of a ribosome-bound nascent chain by NMR spectroscopy. Proc. Natl. Acad. Sci. USA, 104, 16516-16521]. The unfolding of ddFLN5 induced by high concentrations of urea shows a low population of a folding intermediate, as inferred from an intensity-based analysis, a finding that differs from that of ddFLN5 as a ribosome-bound nascent chain. These results suggest that interesting differences in detail may exist between the structure of the domain in isolation and when linked to the ribosome and between protein folding in vitro and the folding of a nascent chain as it emerges from the ribosome. 相似文献
8.
Cycloheximide-ribosome interactions from sensitive and resistant organisms were studied by proton magnetic resonance spectroscopic techniques. The two methyl resonances of cycloheximide upon interaction with ribosomes from Saccharomyces cerevisiae showed preferential broadening. Comparison of cycloheximide line broadening as effected by ribosomes from S. cerevisiae (sensitive) and Microsporum canis (resistant) revealed that less cycloheximide is bound to the M. canis ribosomes. From the decrease in line broadening observed with increasing temperature it may be concluded that cycloheximide-ribosome interaction is a fast exchange reaction. Tetracycline did not compete with cycloheximide for binding site(s) on the ribosomes of S. cerevisiae. 相似文献
9.
Interactions of proteins with small molecules or other macromolecules play key roles in many biological processes and in drug
action, and NMR is an excellent tool for their structural characterization. Frequently, however, line broadening due to intermediate
exchange completely eliminates the signals needed for measuring specific intermolecular NOEs. This limits the use of NMR for
detailed structural studies in such kinetic situations. Here we show that an optimally chosen excess of ligand over protein
can reduce the extent of line broadening for both the ligand and the protein. This makes observation of ligand resonances
possible but reduces the size of the measurable NOEs due to the residual line broadening and the non-stoichiometric concentrations.
Because the solubility of small molecule drug leads are often limited to high micromolar concentrations, protein concentrations
are restricted to even lower values in the low micromolar range. At these non-stoichiometric concentrations and in the presence
of significant residual line broadening, conventional NOESY experiments very often are not sensitive enough to observe intermolecular
NOEs since the signals inverted by the NOESY preparation pulse sequence relax prior to significant NOE build up. Thus, we
employ methods related to driven NOE spectroscopy to investigate protein–ligand interactions in the intermediate exchange
regime. In this approach, individual protein resonances are selectively irradiated for up to five seconds to build up measurable
NOEs at the ligand resonances. To enable saturation of individual protein resonances we prepare deuterated protein samples
selectively protonated at a few sites so that the 1D 1H spectrum of the protein is resolved well enough to permit irradiation of individual protein signals, which do not overlap
with the ligand spectrum. This approach is suitable for measuring a sufficiently large number of protein–ligand NOEs that
allow calculation of initial complex structures, suitable for structure-based optimization of primary drug leads obtained
from high-throughput screening. The method was applied to measure individual intermolecular NOEs between the anti-apoptotic
protein Bcl-xL at 25 μM and a “first generation” small-molecule ligand, for which the spectrum is entirely broadened at stoichiometric
concentrations. This approach is general and can also be used to characterize protein–protein or protein–nucleic-acid complexes. 相似文献
10.
A. H. Jafary-Asl S. N. Solanki E. Aarholt C. W. Smith 《Journal of biological physics》1983,11(1):15-22
In the course of work on the interactions of electric and magnetic fields with both living and dead biological materials, it was noticed that certain published dielectrophoretic yield curves for biological cells showed unexplained deviations in the region of 2 kHz. Dielectrophoretic measurements made at frequencies and magnetic fields which satisfied the nuclear magnetic resonance conditions showed sharply resonant features. Dielectric measurements showed small, but sharp, resonances most easily seen in the dielectric loss curves which had a bandwidth of the order of one Hertz and presented at the frequencies which satisfied the magnetic resonance conditions for the ambient magnetic field. Resonances were found corresponding to the frequencies for electron spin resonance and nuclear magnetic resonance for1H,31P,23Na,37Cl and39K. The onset of these resonances occurs at the value of the steady magnetic field strength so that one quantum of magnetic flux (2.07×10?15wb) would link a single biological cell or pair of cells, approximately 1 G (100μT) in the case of a 5-μm yeast cell. The effects of these magnetic resonance conditions on the mean generation time ofE. coli and on the reaction of the enzyme lysozyme with the substrateM. lysodeikticus cells are also shown. 相似文献
11.
F E Evans 《Archives of biochemistry and biophysics》1979,193(1):63-75
The 40-MHz 31P nuclear magnetic resonance (nmr) spectrum of intact HeLa cells contains seven broad peaks with some detectable splittings. The linewidths were significantly broader than for those of cell-free systems such as cell extracts, indicating that the cellular environment is responsible for the unusual line broadening. Resolution of these peaks at 40 MHz is sufficient to make certain assignments and the relaxation parameters of some of the intracellular metabolites have been measured. The spin-lattice relaxation times (T1) ranged from 0.3 s for adenosine triphosphate (ATP) to about 3 s for inorganic phosphate (Pi) and monophosphate compounds. Nuclear Overhauser enhancements (NOE) were induced by proton irradiation with the possible exception of ATP. The relaxation parameters were compared to those of cell-free compounds and in all cases T1 and NOE were smaller for the intracellular metabolites. The relaxation parameters for ATP were affected the most. This behavior was mimicked with mixtures of cell-free metabolites containing paramagnetic ions. The larger change in both T1 and NOE of intracellular ATP could be accounted for by selective binding of paramagnetic ions. This phenomenon also explains some of the line broadening in the cell spectrum especially that of ATP. The spin-spin relaxation times (T2) of P1 and monophosphate compounds as measured by a pulse technique did not account for the observed linewidths. This is due to the presence of chemical shift envelopes arising from pH heterogeneity. All resonances were broader at 146 MHz because of the line broadening by paramagnetic ions and the presence of chemical shift envelopes. Other mechanisms of line broadening may also be significant. There was little difference in resolution of spectra at 40 and 146 MHz. Water proton linewidths and T2 values were measured for HeLa cells and for some minced tissue preparations. The water linewidth in tissue samples was broader than that in the cell suspension. The large linewidths in tissues arise mainly from chemical shift envelopes caused by magnetic field nonuniformity in the tissue samples. There appears to be a small chemical shift envelope from magnetic nonuniformity in HeLa cells as well. The 1H results on envelopes were extrapolated to 31P studies on cells and tissues. Possible methods for reducing linewidths arising from the various proposed broadening mechanisms were discussed. 相似文献
12.
Repressor--operator interaction in the lac operon. II. Observations at the tyrosines and tryptophans 总被引:3,自引:0,他引:3
H Nick K Arndt F Boschelli M A Jarema M Lillis H Sommer P Lu J Sadler 《Journal of molecular biology》1982,161(3):417-438
This paper shows that 19F-nuelear magnetic resonance spectroscopy on 3-fluoro-tyrosine and 5-fluorotryptophan-substituted wild-type lactose operon repressors from Escherichia coli can be used to examine the interactions with lac operator DNA.A survey of inducer and salt concentration effects on the repressor-operator complex is presented. The data lead us to a scheme for the interactions between the repressor, operator and inducer, in both binary and ternary complexes, that accommodate the results published by others.The complex between the tetrameric repressor and one 36 base-pair operator DNA fragment results in the simultaneous broadening of the resonances from all four N-terminal DNA binding domains. The actual contacts made by these binding domains are similar but probably not identical.The binding of the inducer molecule to the tetrameric repressor results in an allosteric change that can be monitored by the increased intensity of the resonances from individual tyrosine residues in the N-terminal binding domain. This increased N-terminal tyrosine resonance intensity in the complex is transmitted to repressor subunits that have not yet bound an inducer molecule. 相似文献
13.
14.
Mechanism of action of polymeric aurintricarboxylic acid, a potent inhibitor of protein--nucleic acid interactions 总被引:1,自引:0,他引:1
The mechanism of inhibition of protein--nucleic acid complex formation by polymeric aurintricarboxylic acid (ATA) was investigated by proton magnetic resonance spectroscopy. The approach was the synthesis of totally deuterated ATA, followed by a 100-MHz proton magnetic resonance study of its interaction with bovine pancreatic ribonuclease A (RNase), a model nucleic acid binding protein. The binding of ATA to RNase elicited chemical shift changes and line broadening in the C(2)--H resonances of histidyl residues 12 and 119, both of which are located in the active site, whereas that of histidyl residue 105, which resides on the exterior of the protein structure, is unaffected. (Histidyl residue 48 is not observed under our conditions except at high pH.) The epsilon-methylene protons of the lysyl side chains were also broadened upon the binding of ATA. Polymeric ATA displaces cytidine 2'-monophosphate and cytidine 3'-monophosphate from the active site of the enzyme as revealed by nuclear magnetic resonance spectroscopy. These observations suggest that the mechanism of action of ATA involves competition between the nucleic acid and the polymeric ATA for binding in the active site of the protein. Electron spin resonance spectroscopy reveals that polymeric ATA is a stable free radical, thus accounting for the major line broadening effect upon binding to protein. This finding may provide a powerful means of probing the nucleic acid binding site of proteins by proton magnetic resonance spectroscopy. 相似文献
15.
Hexacyanochromate ion, (Cr(CN)6)3-, was applied to ribonuclease T1 (RNase T1), which specifically cleaves RNA chains at guanylic acid residues. From kinetic studies, this anion was shown to bind to the active site of RNase T1 as a competitive inhibitor. Therefore, the line broadening effect of NMR resonances due to binding of (Cr(CN)6)3- was analyzed for the mapping of the active site of RNase T1. His-40 C2 proton resonance was significantly broadened, following His-92 C2 proton resonance upon binding of (Cr(CN)6)3-, while His-27 C2 proton resonance did not show any appreciable line broadening. Moreover, from the pH dependence of the line broadening effect, the binding of (Cr(CN)6)3- was shown to be controlled by the ionic state of Glu-58. Based on the present NMR results and x-ray crystal structure, the active site structure of RNase T1 is discussed. 相似文献
16.
Alexandros D. Petropoulos Ekaterini C. Kouvela Agata L. Starosta Daniel N. Wilson George P. Dinos 《Journal of molecular biology》2009,385(4):1179-7280
Azithromycin is a semisynthetic derivative of erythromycin that inhibits bacterial protein synthesis by binding within the peptide exit tunnel of the 50S ribosomal subunit. Nevertheless, there is still debate over what localization is primarily responsible for azithromycin binding and as to how many molecules of the drug actually bind per ribosome. In the present study, kinetic methods and footprinting analysis are coupled together to provide time-resolved details of the azithromycin binding process. It is shown that azithromycin binds to Escherichia coli ribosomes in a two-step process: The first-step involves recognition of azithromycin by the ribosomal machinery and places the drug in a low-affinity site located in the upper part of the exit tunnel. The second step corresponds to the slow formation of a final complex that is both much tighter and more potent in hindering the progression of the nascent peptide through the exit tunnel. Substitution of uracil by cytosine at nucleoside 2609 of 23S rRNA, a base implicated in the high-affinity site, facilitates the shift of azithromycin to this site. In contrast, mutation U754A hardly affects the binding process. Binding of azithromycin to both sites is hindered by high concentrations of Mg2+ ions. Unlike Mg2+ ions, polyamines do not significantly affect drug binding to the low-affinity site but attenuate the formation of the final complex. The low- and high-affinity sites of azithromycin binding are mutually exclusive, which means that one molecule of the drug binds per E. coli ribosome at a time. In contrast, kinetic and binding data indicate that in Deinococcus radiodurans, two molecules of azithromycin bind cooperatively to the ribosome. This finding confirms previous crystallographic results and supports the notion that species-specific structural differences may primarily account for the apparent discrepancies between the antibiotic binding modes obtained for different organisms. 相似文献
17.
Bruce McConnell 《Journal of biomolecular structure & dynamics》2013,31(6):1407-1421
Abstract An examination of the 1H NMR assignments and exchange properties of the amino resonances of the self-complementary tetramer, d(CGCG) was undertaken with regard to buffer effects, transfer of saturation from the water resonance and temperature dependence of amino 1H line shape and chemical shift. The lack of buffer effect on visible exchangeable proton resonances is evidence for the stringent requirement for nucleo-base protonation at pH values below neutrality, which is greatly reduced in the helical state. For this reason, sharp resonances are observed for both Watson-Crick and non-Watson-Crick cytosine amino protons for base-paired regions. Considerations of monomeric exchange mechanisms for the cytosine and guanine amino protons formed the basis for successful assignment and isolation of their resonances in the helical state by presaturation of the water resonance at selected pH values. Preirradiation of the water resonance at pH <6 would isolate the guanine amino 1H resonances of any self-complementary oligonucleotide, to exploit its high sensitivity as a useful proble of helix ? coil premelting. 相似文献
18.
Solid-state NMR of proteins in frozen aqueous solution is a potentially powerful technique in structural biology, especially if it is combined with dynamic nuclear polarization signal enhancement strategies. One concern regarding NMR studies of frozen solution protein samples at low temperatures is that they may have poor linewidths, thus preventing high-resolution studies. To learn more about how the solvent shell composition and temperature affects the protein linewidth, we recorded 1H, 2H, and 13C spectra of ubiquitin in frozen water and frozen glycerol-water solutions at different temperatures. We found that the 13C protein linewidths generally increase with decreasing temperature. This line broadening was found to be inhomogeneous and independent of proton decoupling. In pure water, we observe an abrupt line broadening with the freezing of the bulk solvent, followed by continuous line broadening at lower temperatures. In frozen glycerol-water, we did not observe an abrupt line broadening and the NMR lines were generally narrower than for pure water at the same temperature. 1H and 2H measurements characterizing the dynamics of water that is in exchange with the protein showed that the 13C line broadening is relatively independent from the arrest of isotropic water motions. 相似文献
19.
In Vivo Natural-Abundance C Nuclear Magnetic Resonance Studies of Living Ectomycorrhizal Fungi : Observation of Fatty Acids in Cenococcum graniforme and Hebeloma crustuliniforme
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Natural-abundance 13C nuclear magnetic resonance spectroscopy has been used to study intact mycelia of the ectomycorrhizal fungi Cenococcum graniforme (Ascomycetes) and Hebeloma crustuliniforme (Basidiomycetes). A number of sharp resonances are observed in living fungi. These signals primarily arise from fatty acyl chains and carbohydrate nuclei. The spectra are interpreted in terms of relative concentrations of the major fatty acids present in the fungal triglycerides. The small line width of fatty acids (mainly oleic, linoleic, and palmitic acids) resonances and spin-lattice relaxation time are indicative of fast rotational reorientations and are consequently thought to arise from fatty acyl chains in fat droplets. We were able to locate the site of lipids accumulation within mycelia using light microscopy and histological staining. Many lipid droplets were observed in mycelia of both species.
These results suggest that fatty acids droplets could be involved in carbon storage and metabolism from ectomycorrhizal fungi.
相似文献20.
Phenotypic Suppression in Escherichia coli by Chloramphenicol and Other Reversible Inhibitors of the Ribosome 总被引:1,自引:0,他引:1
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Antibiotics that interfere reversibly with various aspects of ribosomal function (chloramphenicol, tetracycline, erythromycin, and spectinomycin) are shown to antagonize, at barely inhibitory concentrations, the inhibitory effect of low concentrations of streptomycin (SM) on the growth of Escherichia coli. Paradoxically, these compounds can also replace SM in supporting the growth of conditionally SM-dependent mutants. Chloramphenicol produced about as much phenotypic suppression as SM in SM-sensitive strains, but less than that attainable with high concentrations of SM in resistant strains. The antagonism to SM inhibition and the phenotypic suppression appear to be specific for those growth inhibitors that act on the ribosome. Since inhibitors of the 50S subunit of the ribosome (chloramphenicol, erythromycin) are as active as inhibitors of the 30S subunit, it is suggested that phenotypic suppression by borderline concentrations of ribosome inhibitors does not necessarily depend on an alteration of the recognition region of the ribosome. Alternatively, partial inhibition of the ribosomes might change the environment in a way that would influence the frequency of misreading. Phenotypic suppression by a low concentration of SM as well as by chloramphenicol was found to depend on the presence of a trace of the required growth factor. 相似文献