Martin Lovmar

The mechanism of action of macrolides, lincosamides and streptogramin B reveals the nascent peptide exit path in the ribosome.

The macrolide-lincosamide-streptogramin B class (MLS) of antibiotics contains structurally different but functionally similar drugs, that all bind to the 50S ribosomal subunit. It has been suggested that these compounds block the path by which nascent peptides exit the ribosome. We have studied the mechanisms of action of four macrolides (erythromycin, josamycin, spiramycin and telithromycin), one lincosamide (clindamycin) and one streptogramin B (pristinamycin IA). All these MLS drugs cause dissociation of peptidyl-tRNA from the ribosome. Josamycin, spiramycin and clindamycin, that extend to the peptidyl transferase center, cause dissociation of peptidyl-tRNAs containing two, three or four amino acid residues. Erythromycin, which does not reach the peptidyl transferase center, induces dissociation of peptidyl-tRNAs containing six, seven or eight amino acid residues. Pristinamycin IA causes dissociation of peptidyl-tRNAs with six amino acid residues and telithromycin allows polymerisation of nine or ten amino acid residues before peptidyl-tRNA dissociates. Our data, in combination with previous structural information, suggest a common mode of action for all MLS antibiotics, which is modulated by the space available between the peptidyl transferase center and the drug.

The Kinetics of Ribosomal Peptidyl Transfer Revisited

The speed of protein synthesis determines the growth rate of bacteria. Current biochemical estimates of the rate of protein elongation are small and incompatible with the rate of protein elongation in the living cell. With a cell-free system for protein synthesis, optimized for speed and accuracy, we have estimated the rate of peptidyl transfer from a peptidyl-tRNA in P site to a cognate aminoacyl-tRNA in A site at various temperatures. We have found these rates to be much larger than previously measured and fully compatible with the speed of protein elongation for E. coli cells growing in rich medium. We have found large activation enthalpy and small activation entropy for peptidyl transfer, similar to experimental estimates of these parameters for A site analogs of aminoacyl-tRNA. Our work has opened a useful kinetic window for biochemical studies of protein synthesis, bridging the gap between in vitro and in vivo data on ribosome function.

How initiation factors tune the rate of initiation of protein synthesis in bacteria

The kinetics of initiator transfer RNA (tRNA) interaction with the messenger RNA (mRNA)‐programmed 30S subunit and the rate of 50S subunit docking to the 30S preinitiation complex were measured for different combinations of initiation factors in a cell‐free Escherichia coli system for protein synthesis with components of high purity. The major results are summarized by a Michaelis–Menten scheme for initiation. All three initiation factors are required for maximal efficiency (kcat/KM) of initiation and for maximal in vivo rate of initiation at normal concentration of initiator tRNA. Spontaneous release of IF3 from the 30S preinitiation complex is required for subunit docking. The presence of initiator tRNA on the 30S subunit greatly increases the rate of 70S ribosome formation by increasing the rate of IF3 dissociation from the 30S subunit and the rate of 50S subunit docking to the IF3‐free 30S preinitiation complex. The reasons why IF1 and IF3 are essential in E. coli are discussed in the light of the present observations.

Rate and accuracy of bacterial protein synthesis revisited.

Our understanding of the accuracy of tRNA selection on the messenger RNA programmed ribosome has recently increased dramatically because of high-resolution crystal structures of the ribosome, cryo-electron microscopy reconstructions of its functional complexes, and fast kinetics experiments. Application of single-molecule spectroscopy with fluorescence resonance energy transfer to studies of tRNA selection by the ribosome has also provided new, albeit controversial, insights. Interestingly, when the fundamental trade-off between rate and accuracy in substrate-selective biosynthetic reactions is taken into account, some aspects of the current models of ribosome function appear strikingly suboptimal in the context of growing bacterial cells.

Kinetics of Macrolide Action THE JOSAMYCIN AND ERYTHROMYCIN CASES

Members of the macrolide class of antibiotics inhibit peptide elongation on the ribosome by binding close to the peptidyltransferase center and blocking the peptide exit tunnel in the large ribosomal subunit. We have studied the modes of action of the macrolides josamycin, with a 16-membered lactone ring, and erythromycin, with a 14-membered lactone ring, in a cell-free mRNA translation system with pure components from Escherichia coli. We have found that the average lifetime on the ribosome is 3 h for josamycin and less than 2 min for erythromycin and that the dissociation constants for josamycin and erythromycin binding to the ribosome are 5.5 and 11 nm, respectively. Josamycin slows down formation of the first peptide bond of a nascent peptide in an amino acid-dependent way and completely inhibits formation of the second or third peptide bond, depending on peptide sequence. Erythromycin allows formation of longer peptide chains before the onset of inhibition. Both drugs stimulate the rate constants for drop-off of peptidyl-tRNA from the ribosome. In the josamycin case, drop-off is much faster than drug dissociation, whereas these rate constants are comparable in the erythromycin case. Therefore, at a saturating drug concentration, synthesis of full-length proteins is completely shut down by josamycin but not by erythromycin. It is likely that the bacterio-toxic effects of the drugs are caused by a combination of inhibition of protein elongation, on the one hand, and depletion of the intracellular pools of aminoacyl-tRNAs available for protein synthesis by drop-off and incomplete peptidyl-tRNA hydrolase activity, on the other hand.

The Molecular Mechanism of Peptide-mediated Erythromycin Resistance

The macrolide antibiotic erythromycin binds at the entrance of the nascent peptide exit tunnel of the large ribosomal subunit and blocks synthesis of peptides longer than between six and eight amino acids. Expression of a short open reading frame in 23 S rRNA encoding five amino acids confers resistance to erythromycin by a mechanism that depends strongly on both the sequence and the length of the peptide. In this work we have used a cell-free system for protein synthesis with components of high purity to clarify the molecular basis of the mechanism. We have found that the nascent resistance peptide interacts with erythromycin and destabilizes its interaction with 23 S rRNA. It is, however, in the termination step when the pentapeptide is removed from the peptidyl-tRNA by a class 1 release factor that erythromycin is ejected from the ribosome with high probability. Synthesis of a hexa- or heptapeptide with the same five N-terminal amino acids neither leads to ejection of erythromycin nor to drug resistance. We propose a structural model for the resistance mechanism, which is supported by docking studies. The rate constants obtained from our biochemical experiments are also used to predict the degree of erythromycin resistance conferred by varying levels of resistance peptide expression in living Escherichia coli cells subjected to varying concentrations of erythromycin. These model predictions are compared with experimental observations from growing bacterial cultures, and excellent agreement is found between theoretical prediction and experimental observation.

Trigger factor binding to ribosomes with nascent peptide chains of varying lengths and sequences.

Trigger factor (TF) is the first protein-folding chaperone to interact with a nascent peptide chain as it emerges from the ribosome. Here, we have used a spin down assay to estimate the affinities for the binding of TF to ribosome nascent chain complexes (RNCs) with peptides of varying lengths and sequences. An in vitro system for protein synthesis assembled from purified Escherichia coli components was used to produce RNCs stalled on truncated mRNAs. The affinity of TF to RNCs exposing RNA polymerase sequences increased with the length of the nascent peptides. TF bound to RNA polymerase RNCs with significantly higher affinity than to inner membrane protein leader peptidase and bacterioopsin RNCs. The latter two RNCs are substrates for signal recognition particle, suggesting complementary affinities of TF and signal recognition particle to nascent peptides targeted for cytoplasm and membrane.

Erythromycin resistance by L4/L22 mutations and resistance masking by drug efflux pump deficiency

We characterized the effects of classical erythromycin resistance mutations in ribosomal proteins L4 and L22 of the large ribosomal subunit on the kinetics of erythromycin binding. Our data are consistent with a mechanism in which the macrolide erythromycin enters and exits the ribosome through the nascent peptide exit tunnel, and suggest that these mutations both impair passive transport through the tunnel and distort the erythromycin‐binding site. The growth‐inhibitory action of erythromycin was characterized for bacterial populations with wild‐type and L22‐mutated ribosomes in drug efflux pump deficient and proficient backgrounds. The L22 mutation conferred reduced erythromycin susceptibility in the drug efflux pump proficient, but not deficient, background. This ‘masking’ of drug resistance by pump deficiency was reproduced by modelling with input data from our biochemical experiments. We discuss the general principles behind the phenomenon of drug resistance ‘masking’, and highlight its potential importance for slowing down the evolution of drug resistance among pathogens.

Identification of enzyme inhibitory mechanisms from steady-state kinetics

Enzyme inhibitors are used in many areas of the life sciences, ranging from basic research to the combat of disease in the clinic. Inhibitors are traditionally characterized by how they affect the steady-state kinetics of enzymes, commonly analyzed on the assumption that enzyme-bound and free substrate molecules are in equilibrium. This assumption, implying that an enzyme-bound substrate molecule has near zero probability to form a product rather than dissociate, is valid only for very inefficient enzymes. When it is relaxed, more complex but also more information-rich steady-state kinetics emerges. Although solutions to the general steady-state kinetics problem exist, they are opaque and have been of limited help to experimentalists. Here we reformulate the steady-state kinetics of enzyme inhibition in terms of new parameters. These allow for assessment of ambiguities of interpretation due to kinetic scheme degeneracy and provide an intuitively simple way to analyze experimental data. We illustrate the method by concrete examples of how to assess scheme degeneracy and obtain experimental estimates of all available rate and equilibrium constants. We suggest simple, complementary experiments that can remove ambiguities and greatly enhance the accuracy of parameter estimation.