5 Inhibitors of protein synthesis Many antimicrobial substances inhibit protein biosynthesis. In most cases the inhibition involved one or other of the events which take place on the ribosomes. Only a few agents inhibit either amino acid activation or the attachment of the activated amino acid to the terminal adenylic acid residue of transfer RNA (tRNA). There are many chemical types to be found among the inhibitors of prolein synthesis, a fa ct which has increased the difficulty of unders tanding the molecular nature of their inhibitory effects. Indeed, while the reaction which is inhibited has been ideIHified with some precision in certain instances, the nature of the molecular interaction between the sensitive site and inhibi tor remains generally elusive. The reason lies in the complexity of the reactions leading to the for mation of correctly sequenced polypeptides on the ribosome and also in the complex. ity of the structure of the ribosome itself. Our intention is to provide an outline of the current knowledge of the steps in protein biosynthesis. More detailed discussion is given to those specific reactions which are blocked by the inhibitors of protein biosynthesis. RIBOSOMES These remarkable organelles are the machines upon which polypeptides are elaborated. There are three main classes of ribosomes identified by their sedimentation coefficients. The 80S ribosomes are apparently confined to eukaryotic cells, while 70S ribosomes are found in both prokaryotic and euk aryotic cells. A unique species of50-55S ribosome found only in mamma· tian mitochondria resembles bacterial ribosomes in functional organization and antibiotic sensitivity. The 80S particle dissociates reversibly into 60S and 405 subunits and the 70S into 505 and 305 subunits as the Mg:2+ concentration of the solution is reduced. Both 80S and 70S ribosomes are composed exclusively of protein and RNA in mass ratios of approximately 50: 50 and 35: 65 respecti vely . There are three distinct species of RNA in most ribosomes, with sedimentation coefficients of 295, 185 and 55 in 80S particles from animal cells, 255, 165 and 55 in 80S particles from plant cells 112 T. J. Franklin et al., Biochemistry of Antimicrobial Action © T. J. Franklin and G. A. Snow 1989 Ribosomes 113 and 23S, 16S and SS in 70S particles; 5SS ribosomes contain two RNA species that sediment a t about 16S and 12S. In common with the 70S ribosomes of fun gal mitochondria, it is doubtful whether 5SS ri bosomes contain SS RNA. The protein composition of ribosomes is impressively complex. The 30S subunit of Escherichia coli ribosomes contains 2 1 proteins ('S' proteins), a nd the 50S subunit 34 proteins ('L' proteins). The amino acid sequences of all of these proteins are now known. In recem years great progress had been made in understanding how the ribosome is constructed. T he leading groups in the field have now converged and a consensus model of the structure is available (Fig. 5. 1). This reveals that the large subunit is somewhat 'armchair shaped'. The small subunit can usefully be regarded as being 'head and shoulders' shaped; in the intact ribosome it is positioned 'face' imo the chair with its 'forehead' region resting on the central protuber­ ance of the large subunit. At the time of writing the locations of 19 of the 21 's' proteins have been mapped and rapid progress is being made in the 50S subunit. [n fu nctional terms a translational and an exit (or secrelOry) domain have been defined. The translational domain is ocatedl in the head/shoulder region of the small subunit and the upper part of the armchair. The topographies of the various elongation and initiation factor binding sites are FigureS.' Summary map of protein, RNA and functional sites on the ribosome. lightly shaded sites are located on the far side of the subunits. The letters P, M and E represent th e peptidyltransferase site, the membrane binding site and the nascent protein exit site. The letters 5 and l refer to small and large subunit protein and 1655', 2353', etc. refer t o the 5' and 3' ends respectively of the ribosomal RNAs. Reproduced, with permiss ion, from Annu. Rev. Biochem., S4 (1985) by Annual Reviews nc.I 114 Inhibitors of protein synthesis well understood and represent the culmination of many years of effort by a number of groups using highly sophisticated techniques. Recent work has also identified the ex it domain's location. It is opposite the translational domain on the lower pa rt of the large ribosomal subunit. Once these features had been defined in prokaryotic systems, very similar features were recognized in eukaryotic ribosomes. Further, eukaryotic ribo­ somes bind to rough endoplasmic reticulum at the exit site. This is in accord with a possible mechanism by which cells export proteins from one cellular compartment to another. As we shall see, several medically important antibiotics owe their selecti ve antimicrobial action to a specific attack on bacterial 70S ribosomes, while the 80S particles are left unaffected. These sensitivity differences should be regarded as being due to different ribosomal domains being defined, in many cases, by several ribosomal proteins. The fact that 80S and 70S ribosomes have different proteins means that there are many domains which are unique to each system. The nature a nd role of the various types of ribosomal RNA are still the subjects of intensive study. There is evidence of a f unctional involvement of 23S RNA in the peptidyl­ transferase reaction. In addition, recent proposals suggest that the coclon­ anticoclon helix could be stabilized by co-axial stacking of one spccific region ofthe 16S ribosomal RNA. Now that primary sequences of various ribosomal RN As a re available, progress has been made in mapping the location of the different RNA regions in the three-dimensional models of the ribosomal subunits. No doubt further details of the functional role of RNA in the ribosome structu re will become apparent which may be relevant to our understanding of how some antibiotics inhibit ribosomal activity. STAGES IN PROTEIN BIOSYNTHESIS formation of aminoacyl-transfer RNA Each amino acid is converted by a specific aminoacyl-tRNA synthetase to an aminoacyladenylate which is stabilized by association with the enzyme: Enzyme ATP + amino acid (aa) ~aa-A MP -Enz + PP i Each amino acid-adenylate-enzyme complex then interacts with an amino acid-specific tRNA to form an aminoacyl-tRNA in which the aminoacyl group is linked to the 3'-Q H ribosyl moiety of the 3' terminal adenosyl group of the tRNA by a highly reactive ester bond. aa-AMP-Enz + t RNA ~ aminoacyl- tRNA + AMP + Enz Stages in protein biosynthesis 115 Initiation The mechanism of initiation has been analysed in de tail. Three protein factors, IFI, IF2 and IF3, loosely associated with the 70S ribosome are concerned with initiation. I F I nhane ces the rate of ternary complex forma­ tion between mRNA, initia tor tRNA and 30S ribosomal subunits, but otherwise appears to have little functional significance. IFI has a role in promoting the dissociation of 70S ribosomes, released from previous rounds of polypeptide synthesis, into 30S and 50S subunits. Factor IF3 then binds to the 30S subunit and i s aso l needed fo r the binding of natural mRNA, although not for the binding of an artifici al messenger such as poly(U). The complex containing the 30S subunit, IF3 and mRNA is j oined by rF2, GTP and the specific initiator IRNA, N-formylmethionyl-tRNAr (fMet-tRNAF), the role of IF2 being to direct the binding of fM et-tRNAF to the specific initiator codon, AUG, site. Once it has functioned in this way, the ejection of IF2 requires the hydrolys is of one molecule ofGTP to GOP and inorganic phosphate, and appears to be associated with a conformational change in the 30S subunit. The next stage involves the detachment ofIF3 in the presence of a 50S subunit to permit 70S particle formation. The association of the 50S and 30S subunits is believed to involve interactions between both protein and RN A hainc s of the respective particles. I ni tiation on 80S ribosomes is thought to resemble that on 70S ribosomes M c except that eukaryotic initiation in vivo uses unformylated Met_tRNA , . In add ition, the roles of the specific eukaryotic protein factors, of which there are at least six, in initiation on 80S ribosomes, are less clearly defined than in the 70S ribosome system. Peptide bond synthesis and chain elongation The conventional view of polypeptide synthesis rests largely on the concept of two distinct sites on the ribosome which are usually called the acceptor (sometimes aminoacyl or A) site and the donor (sometimes peptidyl or P ) site (Fig. 5.2). Recent evidence suggests that there may be an additional third tRNA-binding site, the exi t (or E) site. The acceptor si te is the primary decoding site where the codon of the mRNA first interacts with the anticodon region of the specific aminoacyl-tRNA. In the case offMet-tRNAr , however, binding occurs directly to the donor site. The binding of the next aminoacyl­ tRNA to the acceptor site requires protein factors EFT. and EFT". The unstable factor, EFT", binds GTP and then forms a ternary complex with aminoacyl-tRNA. This complex binds to the acceptor site, with accompany­ ing hydrolysis of one molecule ofGTP. GTP hydrolysis is not essential for the binding ofaminoacyl-tRNA, but in its absence the bound aminoacyl-tRNA is not available for peptide bond formation. The role of the stable fa ctor, EFTs, t 1.1 1<2 T ! I I J m RNA ::t:NAr ~ ,~ D GDPtf. f'tIOses IfrmlOitGr t GTP I TOO, P~ide bOnd IQrmf tion , ~"' ~~,..: io n T Fi gure 5.2 Diagrammatic scheme of major steps in polypeptide formation on a 70S ribosome.