Elongation factors on the ribosome

The ribosome is a complex macromolecular assembly capable of translating mRNA sequence into amino acid sequence. The adaptor molecule of translation is tRNA, but the delivery of aminoacyl-tRNAs--the primary substrate of the ribosome--relies on the formation of a ternary complex with elongation factor Tu (EF-Tu) and GTP. Likewise, elongation factor G (EF-G) is required to reset the elongation cycle through the translocation of tRNAs. Recent structures and biochemical data on ribosomes in complex with the ternary complex or EF-G have shed light on the mode of action of the elongation factors, and how this interplays with the state of tRNAs and the ribosome. A model emerges of the specific routes of conformational changes mediated by tRNA and the ribosome that trigger the GTPase activity of the elongation factors on the ribosome.     

Rate limitations in the elongation working cycle and the action mechanisms of GTP-complexed elongation protein factors

Kinetic aspects of the peptide chain elongation process, proper role and the working mechanisms of the GTP-complexed protein elongation factors are discussed. High rates of the codon-dependent binding of aminoacyl-tRNA and translocation are shown to need the mutually exclusive properties of the ribosomal A centre which in the absence of some additional events seems to be unable to possess simultaneously these properties. A centre of translating ribosome is postulated to have a character of dynamic structure providing unsimultaneous consecutive optimization of the aminoacyl-tRNA binding and translocation conditions in accordance with the principle "either binding or translocation". According to this suggestion the rate of elongation is limited by the rate of reversible changes of the A centre structure fitting into the scheme A in equilibrium with B. Each step of this scheme is specifically promoted by corresponding GTP-complexed protein factor. Thus, elongation factors are suggested to be specific modulators of the A centre affinity for the codon-appropriate tRNA and to play a role of complex ligands carrying out an allosteric regulation of the ribosomal functional activity.     

Translocation mechanism of ribosomes

The paper summarizes studies of the molecular mechanism of the dynamic function of the ribosome, i. e. translocation, performed in the author's laboratory during the past decade. The hypothesis of the locking-unlocking of the ribosomal subparticles and the kinematical model of the working ribosome, the processes of spontaneous (factor-free) and factor-dependent translocation, the sequence of events in the factor-dependent translocation, the energetics of translocation and the contribution of the elongation factors with GTP are considered. The following conclusions are made: (1) the translocation mechanism is intrinsic to the structural organization of the ribosome itself but not introduced by the protein elongation factors; (2) the transpeptidation reaction is one of the sources of energy for the work of the translocation mechanism; (3) the protein elongation factors with GTP impart additional energy to the ribosome, including that for translocation, and thus ensure excess power which is realized, in particular, in the increase of the translocation rate and its resistance against inhibitors and hindrances; (4) the promoting role of the elongation factors with GTP does not proceed by a direct conjugation of GTP hydrolysis with translocation, but through the affinity of the elongation factors to the ribosome, with a subsequent compensation of the affinity at the expense of GTP cleavage.     

Alpha-amanitin blocks translocation by human RNA polymerase II

Our laboratory has developed methods for transient state kinetic analysis of human RNA polymerase II elongation. In these studies, multiple conformations of the RNA polymerase II elongation complex were revealed by their distinct elongation potential and differing dependence on nucleoside triphosphate substrate. Among these are conformations that appear to correspond to different translocation states of the DNA template and RNA-DNA hybrid. Using alpha-amanitin as a dynamic probe of the RNA polymerase II mechanism, we show that the most highly poised conformation of the elongation complex, which we interpreted previously as the posttranslocated state, is selectively resistant to inhibition with alpha-amanitin. Because initially resistant elongation complexes form only a single phosphodiester bond before being rendered inactive in the following bond addition cycle, alpha-amanitin inhibits elongation at each translocation step.     

Distinct Conformations of a Putative Translocation Element in Poliovirus Polymerase

The mechanism whereby RNA is translocated by the single subunit viral RNA-dependent RNA polymerases is not yet understood. These enzymes lack homologs of the “O-helix” structures and associated fingers domain movements thought to be responsible for translocation in many DNA-templated polymerases. The structures of multiple picornavirus polymerase elongation complexes suggest that these enzymes use a different molecular mechanism where translocation is not strongly coupled to the opening of the active site following catalysis. Here we present the 2.0- to 2.6-Å-resolution crystal structures and biochemical data for 12 poliovirus polymerase mutants that together show how proper enzyme functions and translocation activity requires conformational flexibility of a loop sequence in the palm domain B-motif. Within the loop, the Ser288-Gly289-Cys290 sequence is shown to play a major role in the catalytic cycle based on RNA binding, processive elongation activity, and single nucleotide incorporation assays. The structures show that Ser288 forms a key hydrogen bond with Asp238, the backbone flexibility of Gly289 is required for translocation competency, and Cys290 modulates the overall elongation activity of the enzyme. Some conformations of the loop represent likely intermediates on the way to forming the catalytically competent closed active site, while others are consistent with a role in promoting translocation of the nascent base pair out of the active site. The loop structure and key residues surrounding it are highly conserved, suggesting that the structural dynamics we observe in poliovirus 3D^pol are a common feature of viral RNA-dependent RNA polymerases.     

Eukaryotic protein elongation factors

In eukaryotes, peptide chain elongation is mediated by elongation factors EF-1 and EF-2. EF-1 is composed of a nucleotide-binding protein EF-1 alpha, and a nucleotide exchange protein complex, EF-1 beta gamma, while EF-2 catalyses the translocation of peptidyl-tRNA on the ribosome. Elongation factors are highly conserved among different species and may be involved in functions other than protein synthesis, such as organization of the mitotic apparatus, signal transduction, developmental regulation, ageing and transformation. Yeast contains a third factor, EF-3, whose structure and function is not yet well understood.     

Domain IV of elongation factor G from Thermus thermophilus is strictly required for translocation.

Two truncated variants of elongation factor G from Thermus thermophilus with deletion of its domain IV have been constructed and the mutated genes were expressed in Escherichia coli. The truncated factors were produced in a soluble form and retained a high thermostability. It was demonstrated that mutated factors possessed (1) a reduced affinity to the ribosomes with an uncleavable GTP analog and (2) a specific ribosome-dependent GTPase activity. At the same time, in contrast to the wild-type elongation factor G, they were incapable to promote translocation. The conclusions are drawn that (1) domain IV is not involved in the GTPase activity of elongation factor G, (2) it contributes to the binding of elongation factor G with the ribosome and (3) is strictly required for translocation. These results suggest that domain IV might be directly involved in translocation and GTPase activity of the factor is not directly coupled with translocation.     

Dynamics of translation on the ribosome: molecular mechanics of translocation

The translocation step of protein elongation entails a large-scale rearrangement of the tRNA-mRNA-ribosome complex. Recent years have seen major advances in unraveling the mechanism of the process on the molecular level. A number of intermediate states have been defined and, in part, characterized structurally. The article reviews the recent evidence that suggests a dynamic role of the ribosome and its ligands during translocation. The focus is on dynamic aspects of tRNA movement and on the role of elongation factor G and GTP hydrolysis in translocation catalysis. The significance of structural changes of the ribosome induced by elongation factor G as well the role of ribosomal RNA are addressed. A functional model of elongation factor G as a motor protein driven by GTP hydrolysis is discussed.     

Possible evolution of factors involved in protein biosynthesis.

The elongation factors of protein biosynthesis are well preserved through out evolution. They catalyze the elongation phase of protein biosynthesis, where on the ribosome amino acids are added one at a time to a growing peptide according to the genetic information transcribed into mRNA. Elongation factor Tu (EF-Tu) provides the binding of aminoacylated tRNA to the ribosome and protects the aminoester bond against hydrolysis until a correct match between the codon on mRNA and the anticodon on tRNA can be achieved. Elongation factor G (EF-G) supports the translocation of tRNAs and of mRNA on the ribosome so that a new codon can be exposed for decoding. Both these factors are GTP binding proteins, and as such exist in an active form with GTP and an inactive form with GDP bound to the nucleotide binding domain. Elongation factor Ts (EF-Ts) will catalyze the exchange of nucleotide on EF-Tu. This review describes structural work on EF-Tu performed in our laboratory over the last eight years. The structural results provide a rather complete picture of the major structural forms of EF-Tu, including the so called ternary complex of aa-tRNA:EF-Tu:GTP. The structural comparison of this ternary complex with the structure of EF-G:GDP displays an unexpected macromolecular mimicry, where three domains of EF-G mimick the shape of the tRNA in the ternary complex. This observation has initiated much speculation on the evolution of all factors involved in protein synthesis, as well as on the details of the ribosomal function in one part of elongation.     

Movement of the decoding region of the 16 S ribosomal RNA accompanies tRNA translocation

The ribosome undergoes pronounced periodic conformational changes during protein synthesis. Of particular importance are those occurring around the decoding site, the region of the 16 S rRNA interacting with the mRNA-(tRNA)(2) complex. We have incorporated structural information from X-ray crystallography and nuclear magnetic resonance into cryo-electron microscopic maps of ribosomal complexes designed to capture structural changes at the translocation step of the polypeptide elongation cycle. The A-site region of the decoding site actively participates in the translocation of the tRNA from the A to the P-site upon GTP hydrolysis by elongation factor G, shifting approximately 8 A toward the P-site. This implies that elongation factor G actively pushes both the decoding site and the mRNA/tRNA complex during translocation.     

Ribosome as a molecular machine

General principles of structure and function of the ribosome are surveyed, and the translating ribosome is regarded as a molecular conveying machine. Two coupled conveying processes, the passing of compact tRNA globules and the drawing of linear mRNA chain through intraribosomal channel, are considered driven by discrete acts of translocation during translation. Instead of mechanical transmission mechanisms and power-stroke 'motors', thermal motion and chemically induced changes in affinities of ribosomal binding sites for their ligands (tRNAs, mRNA, elongation factors) are proposed to underlie all the directional movements within the ribosomal complex. The GTP-dependent catalysis of conformational transitions by elongation factors during translation is also discussed.     

Two crystal structures demonstrate large conformational changes in the eukaryotic ribosomal translocase

Two crystal structures of yeast translation elongation factor 2 (eEF2) were determined: the apo form at 2.9 A resolution and eEF2 in the presence of the translocation inhibitor sordarin at 2.1 A resolution. The overall conformation of apo eEF2 is similar to that of its prokaryotic homolog elongation factor G (EF-G) in complex with GDP. Upon sordarin binding, the three tRNA-mimicking C-terminal domains undergo substantial conformational changes, while the three N-terminal domains containing the nucleotide-binding site form an almost rigid unit. The conformation of eEF2 in complex with sordarin is entirely different from known conformations observed in crystal structures of EF-G or from cryo-EM studies of EF-G-70S complexes. The domain rearrangements induced by sordarin binding and the highly ordered drug-binding site observed in the eEF2-sordarin structure provide a high-resolution structural basis for the mechanism of sordarin inhibition. The two structures also emphasize the dynamic nature of the ribosomal translocase.     

The two main states of the elongating ribosome and the role of the alpha-sarcin stem-loop structure of 23S RNA.

According to the allosteric three-site model of the elongation cycle the ribosome oscillates between two main-functional states, viz the pre-translocational state with occupied A and P sites (E site with low affinity) and the post-translocational state with occupied P and E sites (A site with low affinity). This proposition could be confirmed by a determination of the thermodynamic parameters. High activation-energy barriers were found between both states, namely about 90 kJ mol-1 at 15 mM Mg2+ for either transition (post----pre transition = A-site binding and pre----post transition = translocation). The various A-site states (binding of ternary complex, EF-Tu dependent GTP cleavage, peptide-bond formation) are not separated by significant activation-energy barriers. The rate-limiting step of the elongation cycle is A-site binding, and not translocation as assumed previously. The principal role of both elongation factors is the reduction of the respective activation-energy barrier, thus accelerating the rate of the elongation cycle by several orders of magnitude. Cleavage of a single phosphodiester bond after G2661 of 23S rRNA by the RNase alpha-sarcin abolishes the functions of both elongation factors on the ribosome. This observation implies that the alpha-sarcin stem-loop structure plays an important role in the ribosomal conformational changes involved in the allosteric transitions. Indeed we could demonstrate that suitable oligodeoxynucleotide probes complementary to the alpha-sarcin region induce a conformational change in the 50S subunits; this conformational change causes an irreversible dissociation of tightly coupled ribosomes upon sucrose-gradient centrifugation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Domain III of elongation factor G from Thermus thermophilus is essential for induction of GTP hydrolysis on the ribosome

Two elongation factors (EF) EF-Tu and EF-G participate in the elongation phase during protein biosynthesis on the ribosome. Their functional cycles depend on GTP binding and its hydrolysis. The EF-Tu complexed with GTP and aminoacyl-tRNA delivers tRNA to the ribosome, whereas EF-G stimulates translocation, a process in which tRNA and mRNA movements occur in the ribosome. In the present paper we report that: (a) intrinsic GTPase activity of EF-G is influenced by excision of its domain III; (b) the EF-G lacking domain III has a 10(3)-fold decreased GTPase activity on the ribosome, whereas its affinity for GTP is slightly decreased; and (c) the truncated EF-G does not stimulate translocation despite the physical presence of domain IV, which is also very important for translocation. By contrast, the interactions of the truncated factor with GDP and fusidic acid-dependent binding of EF-G.GDP complex to the ribosome are not influenced. These findings indicate an essential contribution of domain III to activation of GTP hydrolysis. These results also suggest conformational changes of the EF-G molecule in the course of its interaction with the ribosome that might be induced by GTP binding and hydrolysis.     

Inhibition of protein synthesis in vitro by crotins and ricin. Effect on the steps of peptide chain elongation.

The effects of crotin I and crotin II on the partial reactions of polypeptide chain elongation were investigated and compared with the known effects of ricin. Crotin II was a more powerful inhibitor than crotin I, but no qualitative differences between the two crotins were found. Rat liver ribosomes, preincubated with crotins and washed through sucrose gradients, remained inactive in protein synthesis. Among the individual steps of elongation, the peptidyltransferase reaction was unaffected by crotins, but some of the reactions that involve the interaction of elongation factors 1 and 2 with ribosomes were modified. A strong inhibition of the binding of elongation factor 2 to ribosomes and a stimulation of the elongation factor2-dependent GTP hydrolysis were observed; this indicates the formation of a very unstable elongation factor 2--GDP--ribosome complex, which, however, allows a single round of translocation to take place in the presence ofelongation factor 2 and added GTP. The elongation factor 1-dependent GTP hydrolysis was inhibited by crotins, whereas the enzymic binding of aminoacyl-tRNA, to both rat liver and Artemia salina ribosomes, was scarcely affected. In a protein-synthesizing system the inhibition by crotins and by ricin leads to a block of the nascent peptides on the ribosomal aminoacyl-tRNA site, an effect consistent with inhibition at the level of translocation. The mechanism of action of crotins appears to be very similar to that of ricin.