Translation attenuation regulation of chloramphenicol resistance in bacteria — a review

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 Cm^R 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.     

Carbon-13 nuclear magnetic resonance spectroscopy of high-spin iron(III) prophyrin compounds

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.     

A conserved chloramphenicol binding site at the entrance to the ribosomal peptide exit tunnel

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.     

Phosphorus NMR spectra of natural DNA fragments in the course of the B-to-A conformational transition

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.     

Differentiation of the binding of two ligands to a tetrameric protein with a single symmetric active site by 19F NMR

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.     

Structure, Dynamics and Folding of an Immunoglobulin Domain of the Gelation Factor (ABP-120) from Dictyostelium discoideum

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.     

Proton magnetic resonance study of cycloheximide-ribosome interactions

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.     

Identification of individual protein–ligand NOEs in the limit of intermediate exchange

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.     

Dielectric measurements on live biological materials under magnetic resonance conditions

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.     

31P nuclear magnetic resonance studies on relaxation parameters and line broadening of intracellular metabolites of HeLa cells.

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 (P_i) 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.     

Repressor--operator interaction in the lac operon. II. Observations at the tyrosines and tryptophans

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.     

Mechanism of action of polymeric aurintricarboxylic acid, a potent inhibitor of protein--nucleic acid interactions

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.     

Hexacyanochromate ion as a paramagnetic anion probe for active sites of enzymes

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.     

Time-Resolved Binding of Azithromycin to Escherichia coli Ribosomes

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.     

The Amino 1H Resonances of Oligonucleotide Helices: d(CGCG)

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.     

Protein Linewidth and Solvent Dynamics in Frozen Solution NMR

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.     

In Vivo Natural-Abundance C Nuclear Magnetic Resonance Studies of Living Ectomycorrhizal Fungi : Observation of Fatty Acids in Cenococcum graniforme and Hebeloma crustuliniforme

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.

     

Phenotypic Suppression in Escherichia coli by Chloramphenicol and Other Reversible Inhibitors of the Ribosome

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.