Versatile Implementations of an Improved Cell-Free System for Protein Biosynthesis : Functional and structural studies of ribosomal protein L11 and class II release factor RF3. Novel biotechnological approach for continuous protein biosynthesis

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(174) LIST OF STUDIES. The following list presents the work that has been done if forms of short abstracts. For further details, please see the papers enclosed at the end of the thesis. The author’s main contribution Paper I:. The role of the ribosomal protein L11 in translation termination mediated by class I release factors RF1 and RF2 Authors: Bouakaz L., Bouakaz E., Murgola E.J., Ehrenberg M. and Sanyal S. (J. Biol. Chem., 2005 Dec.21, E-published ahead of print). Paper II:. An improved continuous cell-free protein synthesis system using a Sephadex G-25 gel filtration column Authors: Bouakaz L., Katsuda T., Pavlov M. Y., Katoh S., Janson J.C., Ehrenberg M. and Sanyal S. (Manuscript). Paper III:. Functional analysis of Escherichia coli class II release factor RF3 mutants Authors: Zhou Z., Bouakaz L., Sanyal S., Cheng Z., Liu C. W., Ehrenberg M. and Song H. (Manuscript – Short version upon request from our collaborators)). Other publications where the author partially contributed and therefore not mentioned in the thesis Paper IV:. A high-throughput one-step purification method of affinitytagged active Escherichia coli ribosomes Authors: Ederth J., Mandava C. S., Bouakaz L., Dasgupta S., Sanyal S. (Manuscript). Paper V:. Conformation and dynamics of the ribosome stalk protein L12 in solution and on the ribosome Authors: Mulder F.A.A.., Bouakaz L., Lundell A., Venkataramana M., Liljas A., Akke M. and Sanyal S. (Biochemistry 2004, 43, 59305936).

(175) Ne dis pas “Fontaine je ne boirais pas de ton eau”. Le Vieux.

(176) CONTENTS. THE PROTEIN BIOSYNTHESIS IN PROKARYOTES ............................11 The thespians of protein synthesis............................................................11 The ribosome .......................................................................................11 The mRNA ..........................................................................................24 The initiator tRNA...............................................................................24 The initiation factors............................................................................26 The elongation factors .........................................................................31 The tRNAs ...........................................................................................39 The amino acyl-tRNA synthetases ......................................................41 The termination factors........................................................................45 The ribosome recycling factor RRF.....................................................49 The initiation of protein synthesis – Illustrated........................................51 The elongation of protein synthesis – Illustrated .....................................52 The termination of protein synthesis – Illustrated....................................54 The recycling of protein synthesis – Illustrated .......................................56 THE ROLE OF L11 IN TRANSLATION TERMINATION .......................58 BIOTECHNOLOGICAL APPROACH TO HIGH YIELD IN VITRO PROTEIN SYNTHESIS ...............................................................................64 The cell-free system with cellular extracts...............................................64 Cell-free translation system with pure components .................................65 Solid-phase cell-free translation system...................................................66 Continuous cell-free translation systems..................................................66 The CFCF system ................................................................................67 The CECF system ................................................................................67 Reactors ...............................................................................................69 A new generation of continuous cell-free system for protein biosynthesis ..............................................................................................69 FUNCTIONAL ANALYSIS OF Escherichia coli CLASS II RELEASE FACTOR RF3 MUTANTS...........................................................................72 Localization of the interaction site of class II release factor with class I release factors by mutational & biochemical analysis .............................73 ACKNOWLEGEMENTS.............................................................................76 SUMMARY IN SWEDISH – SVENSK SAMMANFATTNING................77 REFERENCES .............................................................................................80.

(177) “Ribosomes, the vital source for protein production…these are the components of the Cell-Free System. Their mission, to boldly go where no other system has ever taken them before”… StarTrek ICM (2000-2005).

(178) ABBREVIATIONS. 30S 50S 70S aa aaRS CECF CFCF CTD DC DNA EC EF fMet GAC GDP GTP IC IF MK NTD PEP PK PPiase PTC RC RF RNA RRF SRL tRNA. The small bacterial ribosomal subunit The large bacterial ribosomal subunit The bacterial ribosome Amino acid Amino acyl-tRNA synthetase Continuous Exchange Cell-Free system Continuous Flow Cell-Free system C-terminal domain Decoding center Deoxyribonucleic acid Elongation complex Elongation Factor Formylated methionine GTPase associated center Guanosine diphosphate Guanosine triphosphate Initiation complex Initiation Factor Myokinase N-terminal domain Phosphoenol pyruvate Pyruvate kinase Inorganic pyrophosphatase Peptidyl transferase center Release complex Release Factor Ribonucleuic acid Ribosome Recycling Factor Sarcin-ricin loop Transfer RNA.

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(180) THE PROTEIN BIOSYNTHESIS IN PROKARYOTES. The thespians of protein synthesis The biosynthesis of proteins in bacteria is a highly controlled and complex process that involves both RNA and protein molecules. In the following paragraphs, I will try to shed some light on the detailed functional and structural interactions of each individual actors of the proteins translation in prokaryotes.. The ribosome The ribosome is responsible for the catalysis of protein synthesis in all the kingdoms of life. It is a complex cellular macromolecule that is involved in the translation of the genetical information held by the messenger RNA (mRNA) into amino acid sequences (proteins). It does so by being the assembly platform on which each codon (nucleotide triplet) of the mRNA is indirectly matched with its corresponding amino acid, through a transfer RNA molecule (tRNA). The tRNA molecule carries the amino acid on one of its ends (acceptor stem) whereas another end (the anticodon stem and loop) is directly involved in the base pairing with the mRNA. The ribosome is an entity constituted of two subunits, one large and one small. It is denoted 70S in prokaryotes as to its relative sedimentation rate (S) and has a molecular mass of 2.4 x 106 Daltons (Da) and a size of ca. 200 Å. The large and small subunits also referred to as 50S and 30S have masses approximated to 1.5 x 106 Da and 0.85 x 106 Da, respectively. The molecular analysis of the ribosome reveals that its functional core is constituted of 67 % RNA and 33 % of proteins (1) (See Figure 1). The high RNA content is attributed some of the ribosome’s main functions, such as the mRNA decoding and the mRNA and tRNA translocation after peptide bond formation (2,3). It was not until the usage of electron microscopes in the 60s that the visualization of the ribosome structure was proposed (Huxley HE, and Zubay G., 1960 JMB 2:10-18). It the following years, further observations revealed the ribosome as an entity constituted of two subunits of different shape and size (4). With 11.

(181) the development of the X-ray crystallography, a high resolution visualization of the full molecule became available at the atomic level (5-7). The constant improvements of the two techniques led to better images of the ribosome which in 2000 resulted in independent revelations of the 50S (8,9) and 30S (10,11) atomic structures. Since then, ribosomal complexes with various translation factors, RNAs and antibiotics became the subject of intense research in the path of better understanding the functional properties of the ribosome and the components it is made of and those it is interacting with.. Figure 1. Illustration of the three dimensional structure of the 70S ribosome. The small picture to the right is the X-ray structure of the 70S ribosome with the 30S subunit in grey (Thermus thermophilus PDB entry 1HR0) and the 50S subunit in black (Haluarcula marismortui, PDB entry 1FFK). The large picture represents the crystal structure of the 70S ribosome from Escherichia coli at 3.5 Å (7), with the 50S subunit in grey and the 30S subunit in blue. The ribosomal RNA is shown in light colors whereas the proteins in this structure have a darker tone.. The structural studies of the ribosome have made it possible to identify specific functional domains on the macromolecule. These regions comprise the mRNA binding site, which is located on the small subunit (along the neck region between the head and the body), the decoding center also on the small subunit, controlling the interaction of the aminoacyl-tRNA and the mRNA, the three tRNA binding sites (E, P and A-sites) with interaction sites on both subunits, the Peptidyl Transferase Center (PTC), the GTPase-associated center (GAC) and the sarcin-ricin binding loop (SRL) and the exit tunnel of the nascent peptide, all located on the 50S large ribosomal subunit. The ribosomal subunit In all known living organisms the ribosome is characterized by two subunits that constitute its entity. The structure of the macromolecule has revealed that it is mainly consisting of RNA. The ribonucleic acid composition is responsible for maintaining its structure, through three identified specific interactions features. The first series are known as the magnesium bridges where the Mg2+ ions form neutralizing bridges between one or more phos12.

(182) phate groups from the secondary structure elements within the sequence. At second the RNA-RNA interactions appear as base pairings between nucleotides associated with secondary structure elements remote in sequence or as interactions between an adenine that inserts its minor groove face into the minor groove of helix (A-minor motifs) which often is a GC pair. The adenine form hydrogen bonds with one or both of the backbone 2’hydroxyl groups of the RNA duplex (12). In third place are the RNA-protein interactions, which involve the sugar-phosphate backbone of the RNA, indicating shape recognition of the ribosomal RNA by the ribosomal proteins. The ribosomal proteins are usually made of at least one globular domain, typically located on the surface of the ribosome and an elongated domain that extends inside the ribosome. In fact, practically all small ribosomal proteins have this structure with the exception of proteins S4 and S15, whereas only half of the large ribosomal proteins share this property. These extensions or tails appear to occupy the space between the RNA helices (13). The 30S small ribosomal subunit The 30S subunit contains 21 proteins allocated around an RNA molecule of 1500 nucleotides called 16S rRNA, for its sedimentation coefficient (See Figure 2). Its atomic resolution was solved at 3-3.3 Å (10,11) and showed that it can be subdivided into four domains; (i) the head, (ii) the neck (mRNA decoding site), (iii) the platform and (iv) the body. These domains are positioned according to their relative connection to the 16S RNA which constitutes the majority of the entity. Accordingly the corresponding secondary-structure of the rRNA constituencies are (i) 5’ domain – body, (ii) the central domain – platform, (iii) the 3’ major domain – head, (iv) the neck which provides a flexible connection between the head and the remaining of the small subunit and (v) the 3’ minor domain (with helices 44 and 45 and the 3’end of the 16S rRNA) (See Figure 3). The small subunit is responsible for the binding of the mRNA as well as the anticodon loop and stem of the incoming tRNA molecules during translation. It is on the 30S that the translation accuracy is believed to occur, a decoding process during which the proper base pairing of codon (mRNA) and anticodon (tRNA) is monitored. The region of the 30S subunit where this occur, also referred to as the Decoding Center or DC (with the universally conserved bases A1492, A1493 and G530) (2,14,15), is mainly constituted of RNA (and ribosomal protein S12) where we find helix 44, the 3’ and 5’ends of the 16S rRNA (10). The rRNA’s 3’end contains the important anti-SD (anti-Shine-Dalgarno) sequence responsible for the base pairing with the mRNA SD sequence. From the crystal structure of the 30S, helix 44 is partially responsible for the proper docking of cognate tRNA, meaning the tRNA with the anticodon loop complementary to the codon sequence on the mRNA, to the A-site (16,17). 13.

(183) Figure 2. Crystal structure of 30s subunit from Thermus thermophilus (PDB entry 1FKA). (A) The small subunit with its 16S rRNA in grey and its 21 proteins in various colors. (B) Crystal structure of the 16S rRNA. (C) Representation of the 21 different proteins that inhabit the small ribosomal subunit. The D-helices are in light blue, whereas the E-sheet structures are in green and the linkers and hinges are in light brown.. Figure 3. Representation of the 30S ribosomal subunit and 16S rRNA crystal structures and the secondary structure diagram of the 16S rRNA from Thermus thermophilus. (A) Tertiary structure of the 30S. The proteins are in color whereas the rRNA is in grey. (B) Tertiary structure of the 16S rRNA. The various domains are colored accordingly. The domain names are those given to the 30S structure found in (A). The head is in blue, the shoulder in red, the platform in yellow, the body in green and the 3’end in grey. (C) Secondary structure diagram of the 16S rRNA. The colored regions correspond to the tertiary structure domains of (B). The names in parenthesis correspond to the 30S regions illustrated in (A) and (B). The central domain is yellow, the 3’major domain is blue and the 3’minor domain is grey. Domain 5 is divided into two sub-domains, the body in green and the shoulder in red.. On the other hand proper base pairing of the tRNA with the mRNA together with the involvement of the 16S rRNA does not appear to be the only way for the ribosome to monitor the decoding fidelity. Additional information about how the ribosome is believed to do that is further emphasized in (3).. 14.

(184) The 50S large ribosomal subunit The large ribosomal subunit is constituted of 34 proteins gathered in a more compact unit, compared with the structure of the 30S subunit. The 50S subunit has two rRNAs with sedimentation coefficients of 5S and 23S, containing 120 and 2900 nucleotides respectively (see Figure 4). The larger rRNA is assigned six secondary structure domains whereas the smaller is commonly designated as the 7th rRNA secondary structure domain (See Figure 5). These 50S subunit have six major interaction domains that are crucial for the catalysis of protein synthesis. Three interaction sites are given to tRNA molecules, which correspond to the different steps of their involvement in translation and designated as the E- for Exit (from where tRNA is leaving the ribosome), P- for Peptidyl (tRNA carrying the polypeptide chain) and Afor Aminoacyl (amino acyl-tRNA entry point) sites. Flanking the tRNA sites, we find the Peptidyl Transferase Center (PTC), where the formation of the peptide bond occurs, the GTPase association center (GAC) where the guanosine nucleotide of various factors is believed to be hydrolyzed and the exit tunnel starting at the PTC and through which the nascent polypeptide chain is believed to come out. The tRNA binding sites The ribosome has three domains for the binding of tRNA molecules which are located in the interface of the two subunits (6). These domains are (i) the A-site, where the aminoacyl- tRNA is accommodated, (ii) the P-site, where the peptidyl-tRNA is bound and (iii) the E-site, where the deacylated tRNA is located and ready to exit the ribosome (See Figure 6). The initiation of protein synthesis commences with the P-site being occupied by the initiator tRNA, brought by initiation factor IF2 in complex with GTP. But during translation elongation, the P-site is inhabited by the tRNA carrying the polypeptide. In both cases the P-site tRNA has its anticodon matching the mRNA codon on the 30S ribosomal subunit and its 3’CCA end in the peptidyl transferase center (PTC). On the other hand, the A-site is the binding site for the new incoming elongator tRNA or aminoacyl-tRNA. It is brought to the ribosome in a ternary complex with elongation factor EF-Tu and GTP. The aa-tRNA recognition by the ribosomal A-site is a crucial step in translation. The ribosomal control of the aa-tRNA anticodon association with the mRNA codon is performed by the A-site decoding center (DC), an RNA structure of the 30S ribosomal subunit.. 15.

(185) Figure 4. Crystal structure of 50s subunit from Deinococcus radiodurans (PDB entry 1NKW). (A) The large subunit with its 23S and 5S rRNA in grey and its 33 proteins in color. (B) Crystal structure of the 23S and 5S rRNA without proteins. (C) Representation of the 33 different proteins that inhabit the large ribosomal subunit. The D-helices are in light blue, whereas the E-sheet structures are in green and the linkers and hinges are in light brown.. Figure 5. Representation of the crystal structures of the 50S large ribosomal subunit and the 23S rRNA and the secondary structure diagram of the 23S rRNA from Haloarcula marismortui. (A) Space filling model of the large ribosomal subunit. The 23S rRNA is colored in red/white and the proteins in blue. In cyan are the L1 and L11 proteins which structures have been individually solved and positioned accordingly. (B) Three dimensional structure of the 23S and 5S rRNA. Each domain is assigned a specific color. Domains I is blue, domain II is light blue, domain III is green, domain IV is yellow, domain V is red, domain VI is purple and the 5S rRNA is cyan. (C) Secondary structure of the 23S and 5S rRNA colored according to the tertiary structure in (B).. 16.

(186) The proper binding of the aa-tRNA induces the hydrolysis of GTP on the EFTu molecule. The elongation factor dissociates from the ribosome which induces a conformational change of the aa-tRNA within the 50S subunit towards the PTC for the subsequent peptide bond formation. After the peptide bond formation the presence of a deacylated- tRNA in the P-site and the peptidyltRNA in the A-site becomes the substrate for the elongation factor EF-G which together with GDP binds to the ribosomal factor binding site inducing a structural twist of the ribosomal subunits. This conformation twist places the tRNA molecules in hybrid P/E and A/P states (Assuming that GDPNP is in fact an analogue of the GDP and not GTP). The exchange of GDP to GTP on the ribosomal pre-translocation complex (the EF-G GEF or G-nucleotide exchange factor) provokes the translocation of the amino acyl-tRNA to the P-site and the deacylated tRNA to the E-site. Subsequently, the hydrolysis of the GTP molecule contributes to EF-G loosing its binding affinity to the ribosome complex which results in its dissociation. The ribosome twist back to its original position with an empty A-site and the peptidyl- and deacylated-tRNA molecules, which are know positioned in the P- and E- site, respectively (18). Exactly how the structural mechanisms of translocation work still remains an open issue. The mechanism of E-site tRNA molecule release is still an ambiguous matter, even though recent work by Nierhaus et al. have indicated the requirement of a +1 frameshift for the E-site tRNA to release from its codonanticodon interaction with the mRNA (19). Additionally, the binding of the ternary complex to the A-site for a new round of elongation induces the release of the E-site tRNA, attributing this function to the 30S ribosomal subunit. In particular, this has been shown to occur during the post-codon recognition phase and prior to GTP hydrolysis on EF-Tu (20). The ribosomal protein L1 has also been suggested to participate in the release mechanism of the Esite tRNA, through the movement of the L1-stalk induced by the twist-like rotation of the ribosomal subunits observed during the elongation process (9). The Decoding Center (DC) The decoding center is situated in the interface between the two ribosomal subunits within the A-site (10). This region where the codon and anticodon pair, is composed of four domains: the head, shoulder, platform and helix 44 (See Figure 7). The particularity of the decoding center is that specific bases of the 16S rRNA, nucleotides A1492, A1493 of Helix 44 and the shoulder nucleotide G530, closely interact with the first two positions of the codonanticodon pair upon cognate fit. The tRNA and mRNA ribose-phosphate backbone take a spatial geometry that favors the hydrogen bonding with nucleotides A1492 and A1493 which flip out of the helix 44. Additionally the binding of the tRNA molecule induces a rotation of the shoulder nucleotide G530, further supporting the hydrogen bond of the helix 44 nucleotides (3,16,21) (See Figure 7). 17.

(187) Figure 6. Representation of the three-dimensional Cryo-EM with three tRNA molecules at their respective binding sites during protein synthesis. The splitting of the 70S ribosome into its subunits 30S in blue and 50S in silver interface sides. The E-site deacylated tRNA, the Psite bound peptidyl-tRNA, and the A-site bound aminoacyl tRNA are colored in orange, dark blue and green respectively.. Figure 7. Illustration of the decoding site at the 30S ribosomal subunit interface. The 16S helix 44 and its nucleotides are shown in light brown. The flipping of the nucleotide A1492, A1493 and G530 is shown by arrows. The cognate mRNA codon is shown in purple and the cognate tRNA anticodon is shown in green. The numberings used are for Escherichia coli rRNA. The picture was adopted and modified from (22).. In turn, all these conformational changes initiate a rearrangement of several proteins of the 50S ribosomal subunit through the tRNA 3’ end induced fit in the PTC. This reconfiguration of the large subunit structure is believed to be the signal for the hydrolysis of the GTP molecule of elongation factor EF-Tu and the subsequent peptide bond formation. The Peptidyl Transferase Center (PTC) The peptidyl transferase center is located in the 50S large ribosomal subunit at the bottom of the cleft below the central protuberance interface side and mostly constituted of RNA (8,9,23) (See Figure 8A). The region of the 23S 18.

(188) rRNA structure that defines the activity of PTC is the central loop of domain V, also identified as the “PTC ring” which is the junction of five helices (2426) (See Figure 8B). This structure, highly enriched with modified nucleotide residues, has the utmost percentage of universally conserved residues. Some of them have been shown to play a crucial role in the catalytic activity of the PTC (27-30) (see Table 1). Even though no ribosomal protein has been identified within at least 15 Å of the PTC, their involvement in sustaining the catalytic site conformation cannot be disregarded (31,32). The PTC is involved in the catalysis of two major events in protein synthesis: the formation of peptide bonds during elongation and the peptide bond hydrolysis during translation termination. The formation of the peptide bond involves the nucleophilic attack of the A-site aminoacyl-tRNA Damino group (-NH2) on the carboxyl group (COO-) at the 3’ end ribose of the P-site peptidyl-tRNA, probably with the help of the N3 and ribose 2’-OH of the 23S rRNA nucleotide A2451. This generates a transition structure called the tetrahedral intermediate, which decays rapidly leading to a proton donation to the 3’end oxygen group of the newly deacylated P-site tRNA and results in a deacylated tRNA in the P-site and an elongated by one amino acid peptidyl tRNA in the A-site (See Figure 9).. Figure 8. The peptidyl transferase center and interactions leading to peptide bond formation. (A) Structure of the 50S ribosomal subunit and the location of the peptidyl transferase center, illustrated by a white circle. (B) Secondary structure of the 23S rRNA domain V illustrating the PTC ring. Some of the nucleotides that are involved in the catalytic activity of the site are represented by their Escherichia coli numbers. (C) Illustration of the interaction of the 3’ CCA ends of the P-site and A-site tRNA molecules with the PTC.. The reaction is made possible by the positioning of the tRNA molecules inside the 50S ribosomal subunit interface cleft. Here the 3’ end C74 and C75 nucleotides of the peptidyl-tRNA base-pair to the PTC P-loop residues G2251 and G2252 (33), whereas the 3’ end C75 nucleotide base-pairs with the PTC A-loop residue G2553 (34,35). In addition, the close proximity of 19.

(189) the 23S rRNA nucleotide A2541 suggests it to be the catalytic nucleotide that participates in the proton donation process (See Figure 8C).. Figure 9. Scheme for the acid-base reaction of the peptide bond formation. The D-amino group (-NH2) of the A-site aminoacyl-tRNA attacks the carbon atom of the P-site peptidyltRNA carbonyl group (-COO) forming the so-called tetrahedral transition state which is short leaved. The reaction ends with the generation of a deacylated-tRNA in the P-site and a peptidyl-tRNA in the A-site carrying the extra added amino acid. The amino acid chain, originally on the peptidyl-tRNA is illustrated by grey filled stars, whereas the new amino acid attached to the aminoacyl-tRNA is represented by a white star.. Nevertheless the conformation of its role as the catalytic nucleotide remains to be clarified. In the PTC, the tRNA ends are further stabilized through a Aminor interaction between their A76 nucleotide with the 23S rRNA basepairs U2506-G2583 (P-site tRNA) and U2450-C2501 (A-site tRNA) (12,36,37). The exact chains of chemical events that lead to the release of the polypeptide remain a puzzle that still requires further structural elucidations. What has been established so far is that the hydrolysis of the ester bond might be triggered by the presence of the universally conserved domain III GGQ motif of the class I release factors (See paragraph on release factors) found inside the PTC, a motif that when mutated prevents translation termination (38-42). The second role of the PTC is the release of the polypeptide from the peptidyl-tRNA during the translation termination. This action is mediated by the interaction of the class I release factors with the ribosomal A-site, upon recognition of a stop codon in the DC. Similarly to the peptide bond formation, the reaction of the polypeptide release involves the nucleophilic attack of the ester bond joining the amino acid chain with the P-site bound peptidyltRNA, only this time through the coordination of a water molecule, in a scenario similar to the hydrolysis of the ester bond observed in the absence of release factors and presence of acetone, where the peptide residue is transferred to the alcohol hydroxyl group (38,40,42-48).. 20.

(190) Table 1. Functional activities of some of the 23S rRNA nucleotides of the PTC.. The exit tunnel of the nascent peptide From the heart of the 50S ribosomal subunit, which can be symbolized by the PTC begins a long and narrow tubular like structure extending throughout the large subunit, called the exit tunnel (See Figure 10). It is through this 100Å long tunnel and a diameter of 15Å, that the nascent polypeptide is likely channeled and subsequently released from the ribosome. It is mainly constituted of rRNA (23S rRNA domain I through V) with the exception of a narrow constriction within it, formed by the two large ribosomal proteins L4 and L22 (36). On the other hand the tunnel exit is largely constituted of the ribosomal proteins. These have been shown to participate in post-translational processes, which are out of the scope of this thesis and will therefore not be discussed further.. 21.

(191) The Ribosome Stalk and the GAC The ribosomal domain responsible for the binding of translational factors is called the ribosome stalk region (See Figure 11A). It is composed of the ribosomal proteins L11 and the pentameric complex [L10 x (L7/L12)4]. In Escherichia coli ribosomes, the L7/L12 is the only protein with no interact with the rRNA and found in four copies (49-52). It is composed of two homodimers which are independently connected to the L10 protein Cterminal, between positions 71 and 164 of the last mentioned (52-55). The L7/L12 has itself three major domains: (i) the N-terminal domain, composed of residues 1 to 37, which connect the protein to L10 when dimerized, (ii) the flexible hinge domain with residues 38 to 49, providing the protein with a high movement capability and (iii) the C-terminal domain with residues 50 to 120, which is attributed the function of binding the translational factors and the participation in the catalytic process of their GTP hydrolysis (56-59). A lot of studies have been performed to figuring out the structural rearrangement of the tetrameric (L7/L12)4 protein complex on the ribosome, unfortunately only resulting in many proposed models for the dimerization mode of L7/L12 (52,60-63). More structural studies are required to further elucidate the spatial conformation of the L7/L12 during translation; nevertheless it is clear that its role in the elongation process involves major domain movements within the stalk region (64-67). The protein L10 of the stalk pentameric complex and the L11 protein bind together a specific region of the 23S rRNA domain II (L10 & L11 binding region). The L10 interacts with helices H42 and H43, whereas L11 contacts helices H43 and H44 (residues 1051-1108), also known as the GTPase associated center or GAC. The L11 ribosomal protein is a highly conserved ribosomal protein located at the base of the L7/L12 stalk. The L11 is composed of a flexible Nterminal domain, that is believed to interact with the GTP binding domain of the translation factors and a C-terminal domain which is tightly connected to the 23S rRNA via the interaction with helices H43 and H44 of the GAC (64) (See Figure 11B). The GTPase-associated center and the helix 95 (H95) or sarcin-ricin loop (SRL) of the 23S rRNA domain VI have together been functionally assigned the role of binding all the translational GTPases and promoting the hydrolysis of their GTP in the course of their action (59,68,69). These two universally conserved regions of the ribosome have been shown to change the spatial orientations between them during the different steps of protein biosynthesis. What has been observe is a contact of the SRL A2660 residue region with the G-domain of EF-Tu and domain I of EF-G (70-74) and an interaction of the GAC A1067 residue (from H43) with domain V of EF-G and either the tRNA “elbow” or domain III of EF-Tu (70,74-76). Upon factor binding, the GAC lobe like structure closes in towards the 50S ribosomal body maintaining thereby perhaps the factor in 22.

(192) place for its chore. Once the task completed, the GAC returns to its original position. During this process the SRL does not appear to change its structural conformation (67).. Figure 10. Illustration of the exit tunnel of the translating ribosome. (A) Cryo-EM structure of the Escherichia coli translating 70S ribosome. The 30S subunits is shown in light brown, the 50S in light blue, the mRNA in orange, P-site tRNA carrying the polypeptide is in green whereas the A-site tRNA in magenta. The polypeptide chain is represented by colored beds and the PTC is highlighted by a white circle. (B) Rough dissection view of the 70S ribosome illustrated in (A) showing the exit tunnel. The exit tunnel is shown by a red thread, the P-site peptidyl tRNA is in green, the mRNA is in magenta and the PTC is highlighted similarly to (A). The 30S and 50S subunits are colored in orange and grey, respectively.. Figure 11. Position of the ribosome stalk base, the GAC and the sarcin-ricin loop on the 50S ribosomal subunit from Thermotoga maritime. (A) Illustration of the 50S with the rRNA is grey and the proteins in light blue. The region of the stalk base is in a grey frame. The 23S rRNA domain of the sarcin-ricin loop is shown is magenta and the domain of the L10 and L11 binding region is shown in light brown. The protein region of the CTD of L11 and NTD of L10 are yellow and blue, respectively. The structure of L7/L12 tetramer remains unsolved; its location is therefore illustrated by a green dashed ellipsoid. (B) Representation of the secondary structure of the L11 CTD and L10 NTD interaction with the helices H42, H43 and H44 of the domain II of the 23S rRNA. The contacts with the 23S rRNA and the L10 are in red/green whereas those of the GAC (H43 and H44) with L11 are in orange/cyan. The picture was adapted from (63) and further modified.. 23.

(193) The “open” to “closed” movement of the GAC after the proper binding of the factors, could theoretically provide the necessary conformational orientation of the factors G-domains to the vicinity of the SRL A2660 residue and hence initiate the GTP hydrolysis sequence. But this remains to be further clarified.. The mRNA The ribosome is the only known entity capable of translating the genetic code carried by the mRNA to fully active proteins in any living cell. To do that it is crucial that the macromolecule recognizes specific sequences (TIR) on the ribonucleic acid chain. In doing so the ribosome slides on the mRNA to the ribosome binding site (RBS), a stretch covering approximately 30 nucleotides and from where the translation commences. How the ribosome really does this is not yet fully understood, but research in this area has so far indicated that the ribosome uses a series of signals for positioning itself accordingly. This recognition is believed to occur at the mRNA 5’ untranslated region (5’ UTR) end, a ribonucleic domain which includes the SD sequence (GGAGG ± 2 nucleotides upstream of the initiation codon AUG or AUU) also known to interact with the 3’end of the 30S ribosomal subunit 16S rRNA (77). At this stage the ribosome positions itself on the mRNA, where the AUG initiator codon is present and capable of receiving the initiator tRNA fMet-tRNA fMet brought by initiation factor IF2. In bacteria the most commonly used initiation codon is AUG. Translation initiation has also been observed on GUG and UUG codons at frequencies of 8 and 1 % respectively in Escherichia coli (78). One interesting common fact for all gram negative bacteria such as Escherichia coli, is the usage of leader sequences, which are pyrimidine-rich, regions regarded as interaction sites of ribosomes as well as the S1 small ribosomal protein (79,80). One could imply from this that the ribosome primary anchoring point on the mRNA is a protein-RNA interaction followed by the sliding and further unwinding of the macromolecule through RNA-RNA interactions to the RBS and the subsequent localization at the initiator codon. Gram positive and archaea bacteria on the other hand, do not have leader sequences and start therefore the initiation at or a few nucleotides 5’ upstream of the initiation codon (81), in a pathway which appears to share similarities with the initiation in eukaryotes (82-85). X-ray crystallographic studies have confirmed that the mRNA helical structure is disrupted to fit in the ribosome, but how this is achieved is yet to be clarified (6,21).. The initiator tRNA This tRNA incorporates the first amino acid to all chains of polypeptide which give rise to fully active proteins once their translation is accomplished by the 24.

(194) ribosome. It always carries a methionine amino acid (Met) that is different from the ones used further in the translation process by being N-formylated, hence the name fMet-tRNA fMet. This restricted modification excludes its usage as elongator tRNA, ensuring thereby the correct initiation of translation at the TIR of any given mRNA. This unique property of the bacterial initiator tRNA has also been found in chloroplasts (plants) and mitochondria (eukaryotes) (86-88). The fMet-tRNA fMet recognizes the AUG initiation codon on the mRNA and localizes itself in the P-site. But as previously described, initiation of translation starting at codons with single base changes relative to this codon has been observed. These codons are also decoded by the initiator tRNA as Nformylated methionine. In addition to the discrepancy of the carried amino acid the initiator tRNA itself is different from all other tRNA molecules in its interaction with the factors involved in elongation. The initiator tRNA is brought to the ribosome by initiation factor IF2 and is not recognized by the elongation factor EF-Tu as are all other tRNAs. This is due to structural features that are only distinguished by IF2 and discriminated by EF-Tu. As seen in Figure 12, the elements are located in the anticodon stem (three conserved GC base pairs), acceptor stem (absence of Watson-Crick base pair between position 1 and 72) and the dihydrouridine (D) stem (the presence of a purine11-pyrimidine-24 in contrast to the pyrimidine-11-purine-24 base pair in other tRNAs) (89). The GC base pairs in the anticodon stem render the structure of the initiator tRNA anticodon loop less flexible, an important feature for its targeting to the ribosomal P-site (90,91). In addition to the differences of tRNA recognition for the proper initiation of translation, one interesting observation is that both the initiating and elongating methionine tRNAs are aminoacylated by the same enzyme, the dimeric methionine synthetase or MetRS. The synthetase scrutinizes only the anticodon for its binding to the tRNA and since it is the same for both types of Met tRNAs, the amino acylation process is identical. On the other hand, the formylation of the initiating methionine amino acid is conducted by the methionyl-tRNA transformylase (MTF) on the amino acylated tRNA. This enzyme catalyzes the transfer a formyl group from N10-formyltetrahydrofolate (THF) molecule to the D-amino group, a process that can only happen in the absence of the 1:72 base pairing in the acceptor stem of the tRNA, rendering the 3’ end of the acceptor arm to be a 5 nucleotide long single strand (92). It is from this point that the two types of methionine-tRNA molecules enter their different pathways in the protein biosynthesis. The formylation of the amino acid has been observed to greatly favor the binding to IF2 leading to the subsequent positioning of the fMet tRNAfMet at the initiation codon where the translation is to initiate (93). The solved structure of the 70S ribosome carrying the initiator tRNA in the P-site revealed that the tRNA interacts with the 30S subunit 16S rRNA at the anticodon stem and loop and with the 50S subunit 23S rRNA at its acceptor arm (CCA end) as well as its D-stem. Additionally a protein-RNA interaction was identified between the large ribosomal protein L23 and the initiator tRNA T-loop (6). 25.

(195) Figure 12. Representation of the structural features that differentiate the initiator tRNA fMettRNA fMet and the elongator tRNA Met-tRNA Met.. The initiation factors Three initiation factors have so far been identified in bacteria. Their exact function in translation is still the subject of intense investigations. Initiation factor IF1 The first initiation factor IF1 is the smallest of three with only a mass of 8300 Da and encoded by the infA gene. The structure of this protein consists of five beta barrel strand with a loop between strands 3 and 4 capping one end of the barrel as determined by NMR spectroscopy (94). IF1 only differentiates itself from the archaeal and eukaryotic homologue by not having a D-helix at the C-terminus (See Figure 13). Interaction studies of IF1 with the 30S ribosomal subunit have indicated that the factor binds in a cleft between the 530 loop, the 16S rRNA helix 44 (residues A1492 and A1493) and the small ribosomal protein S12, a position that strongly corresponds to the Asite tRNA (95-97). This could suggest that IF1 would somehow help stabilize the binding of binary complex of fMet- tRNAfMet and IF2 on the 30S, by preventing other tRNAs to bind until the subunits are properly joined and ready to start the elongation process. The constant pursuit of finding the role of IF1 in translation initiation still is the subject of many ongoing investigations. Our group in particular, led by my Professor Ehrenberg M. and my colleagues, Antoun A., Pavlov M. Y. and Lovmar M. recently obtained data indicating that IF1 enhances the ability of IF3 in preventing the formation of initiator tRNA-less 70S ribosomes (submitted manuscript). Additionally, IF3 and IF1 appear to collaborate on 26.

(196) the small ribosomal subunit to insure the fast dissociation of non-initiator tRNA molecules on the 30S ribosomal subunit, implying that IF1 plays a considerable role in the accuracy of initiator tRNA selection (submitted manuscript).. Figure 13. Representation of the structure of initiation factor IF1 from Escherichia coli (PDB entry 1AH9) and its homologues: Human eIF1A (residues 40-125 with PDB entry 1D7Q), archeae aIF1A from Methanococcus jannaschii (PDB entry 1JT8) and the cold shock protein A (CspA) from E. coli (PBD entry 1MJC).. Initiation factor IF2 The second initiation factor IF2 is the biggest and is encoded by the infB gene. This gene is part of the polycistronic nusA operon that contains additional genes expressing other protein.. Figure 14. Representation of the primary structure of the initiation factor IF2 from Escherichia coli. The domain boundaries and the length of the three isoforms of IF2 are indicated below the picture. The structures have the PDB entries 1ND9 and 1G7T respectively.. 27.

(197) In bacteria, initiation factor IF2 is attributed the responsibility of selecting the initiator tRNA until its stabilization on the 30S ribosomal subunit (98), which in turn stimulates the joining of the 50S subunit for the formation of active 70S ready for elongation (99,100). It is the only initiation factor capable of exchanging G-nucleotides, a functionally crucial process (101,102). In Enterobacteriaceae, such as Escherichia coli, three different isoforms of IF2 (IF2-1, IF2-2 and IF2-3) have been identified (103,104). These proteins are translated from three independent but in frame translational start sites from the infB mRNA. The last two mentioned differ from the first by having 157 and 164 less amino acid residues, respectively (105). So far only the Bacillus subtilis is known to contain a single isoform of IF2 (106). Strangely, the presence of all three isoforms of IF2 was shown to be required for optimal cell growth in bacteria. The structure of IF2 is divided into six major domains where the N-terminal region regroups domains I-III and the Cterminal region is conferred into domains IV-VI, with domain VI being additionally partitioned in sub-domains VI-1 and VI-2 (See Figure 14) (107109). The atomic resolution of the Escherichia coli IF2-1 has not yet been determined but from circular dichroism (CD) and NMR data available to this date, the N-terminal domains I to III showed unstructured and flexible regions with substantial helical content and appeared to be connected to the Cterminal domains IV to VI via a linker (109,110). From sequence alignment studies it was found that the C-terminal region of the protein showed a remarkable interspecies homology with its counterpart found in both archaeabacteria (aIF5B) and eukaryotes (eIF5B) (107,111). The GTP binding domain of IF2 has been attributed to the domain IV, which structure shares the highest homology with the G-domains of factors from all known kingdoms. The availability of the crystal structure of the archeon Methanobacterium thermoautotrophicum aIF5B (112), has made it possible to draw exciting functional parallels with the recently solve Cryo-EM density map of Escherichia coli IF2-2 with the GTP analogue GDPNP in complex with initiator tRNA on the 70S ribosome (59).The IF2 occupied a density in the intersubunit space of the 70S ribosome. Direct contacts, with both the 50S and 30S subunits and initiator tRNA were observed. What one must bare in mind here is that the obtained Cryo-EM of IF2 was made from a mixture containing also the other two initiation factors IF1 and IF3, suggesting that the conformation of IF2 represents a step before release of IF1 and IF3, which could very well be different from a complex with IF2 alone. This has recently been suggested in the recent density map of IF2 alone in complex with the 70S ribosome with either GDP or the GTP analogue GDPNP (113). It is clear that isolating the different states of IF2 in the initiation step of translation and hence the identification of the sequence of events that lead to a 70S postinitiation, the end product of initiation of protein synthesis are more difficult than previously anticipated. Nevertheless the progresses made in the elucidation of the factor’s structure and its function is moving forward. Domains I, 28.

(198) II and III of IF2-1, that constitute the factor’s N-terminal region, are highly flexible structures making it difficult to properly characterize their full density by both X-ray and Cryo-EM techniques available to date. Nevertheless previous studies have shown that fragments from domains I and II of Escherichia coli IF2 bind to 30S (99,100). Additionally, the Cryo-EM structure obtained from the IF2-2 in complex with the 70S ribosome, revealed for the first time a density of the N-terminal domain of the factor extending over the surface of the 30S ribosomal subunit (59). The domain IV or G-domain appeared clearly to reside in the same site as that of the elongation factors EFTu and EF-G, indicating the possible existence of a common mechanism by which the ribosome affects the guanosine nucleotide binding translation factors (112). The close proximity of the G-domain to the GTPase association center (GAC), constituted of the sarcin-ricin loop (SRL) of 23S rRNA, 50S L11 protein and the C-terminal domain of L7/L12 stalk suggest that the later one confers a structural change in the region stimulating thereby the GTPase activity of IF2 (65,114). Recent NMR studies performed on the interaction between the eukaryotic initiation factors eIF1A and eIF5B (115), homologues of IF1 and IF2 respectively, together with the cross-linking results reported for the interaction between Escherichia coli IF1 and IF2 (97) suggested domain V of IF2, with helix 8 (H8) as the interaction platform of IF1. Additionally, cross-linking studies of IF2 also showed interactions of domain V residues with the T-arm of the fMet-tRNAfMet placing the domain within the A-site of the 50S ribosomal subunit (116). The interaction of domain VI is attributed to the initiator tRNA via the interaction of its Cterminal (domain VI-2). Amino acid deletion studies showed that domain VI protect the initiator tRNA from spontaneous decylation as well as binding to the ribosome (100). In the Cryo-EM density map by Allen et al. (59) the domain VI-1 of IF2 interacts extensively with the surface of L14, while domain VI-2 contacts a large surface of L16 as well as 23S rRNA bases 2550– 2556 and 2601–2602. Additionally, the initiator tRNA appears to have been lifted in the P-site, a movement which has not been observed in the CryoEM density by Myasnikov et. al. (113) where the IF2 contacts the tRNA well positioned in the P-site via its D-loop and not its CCA end, attributing this effect to the presence of IF3. As a result of this intermediate step, it was proposed that the IF3 possesses a proof-reading mechanism of IF3 to ensure the sole presence of the initiator tRNA in the P-site. The further interaction with IF1 and IF2 would, once IF3 is released re-confer the initiator tRNA is position in the P-site followed by the lost of the interaction with domain VI2, a conformation change that is believed to induce the signaling for the subsequent release of IF1. Initiation factor IF3 The last of the initiation factor is a molecule of about 20.4 kDa with 180 amino acids encoded by the InfC gene, which is in tandem with the genes 29.

(199) encoding the ribosomal protein L35 and L20, by sequential order. Various structural studies have shown that the initiation factor IF3 was made of two structural regions identified as the N-terminal (IF3N) and the C-terminal (IF3C) domains (117,118). These two parts are linked together by a flexible linker rich in lysine amino acids (119,120) (See Figure 15). This initiation factor has been attributed various functions such as a ribosome subunit antiassociation factor (preventing the association of the 50S ribosomal subunit) by supplying the translation initiation pool with free 30S subunit (121-123), accelerating the formation of initiation complexes as well as playing an essential role in the proof-reading mechanism of the initiator tRNA docking at the P-site on the 30S ribosome subunit (124-131). In addition to its role in the initiation of translation, IF3 has also been identified as a key player in the recycling step of protein synthesis, notably in the dissociation of the deacylated tRNA from the post-termination complex P-site followed by the dissociation of the 70S ribosome into subunits (132-134).. Figure 15. Representation of the structure of initiation factor IF3. The IF3 N-terminal domain (IF3N) is from B. stearothermophilus (PDB entry 1TIF) and the IF3 C-terminal domain (IF3C) is from E. coli (PDB entry 2IFE). The side chains of the arginine residues which mutations have shown to affect the binding to the 30S ribosomal subunit are colored in blue with their corresponding number.. Recently two submitted studies from the Ehrenberg group have shed some more light about the interplay between the various initiation factors during the initiation of translation. It was shown that even though IF3 spontaneously dissociates from the 30S pre-initiation due to the presence of IF1, the third initiation factor also prevents much more strongly the docking of the ribosome 50S subunits in the presence of IF1. Additionally the rate of subunit association in the nonattendance of initiator tRNA was practically negligible with both factors there but more significant if IF3 wasn’t. These results propose a mechanism for IF3 as to prevent the accumulation of 70S ribosome without initiator fMettRNAfMet and that the interaction with initiation IF1 enhances this process. In 30.

(200) the second study, the high accuracy of protein synthesis is conferred to the co-action of IF1 and IF3, by reducing the affinity of all tRNAs to the 30S as well as the docking of the 50S subunit, and the preference of IF2 in their presence to specifically select the formylated initiator tRNA over all other tRNAs and binding it the mRNA programmed 30S subunit catalyzing thereby the formation of the 70S pre-initiation complex. When protein synthesis is terminated, the 70S post-termination complex has a deacylated tRNA sitting in the P-site. In order for the ribosome to start another round of initiation it is crucial that the subunits, the mRNA as well as the P-site tRNA dissociate from the ribosomal post-termination complex. This process has been shown to involve three factors, notably elongation factor EF-G, the ribosome recycling factor RRF and initiation factor IF3. In this process EFG and RRF split the ribosome into subunits allowing the IF3 bind the 30S subunit, dissociating the deacylated tRNA from the post-termination complex and preventing thereby the 50S to bind back (132-139). Hence this three-factor-dependent stable dissociation of ribosomes into subunits ends the ribosome translation cycle and provides subunits ready are for the next round of translation.. The elongation factors The initiation of translation ends with the amino acylated initiator tRNA fMet tRNA fMet in the P-site of the ribosome, leaving the A-site empty and ready to accept an elongator tRNA (aa-tRNA), marking the launch of the translation elongation step. The elongation phase is characterized by the constant addition of amino acids corresponding to the specific genetic sequence carried by triplet bases on the mRNA denoted codons. During the maturation of the polypeptide, the mRNA shifts three bases on the ribosome for each incorporated amino acid. This complex scenario is divided into three moments that continue on recycling until a stop codon reaches the Asite. The first moment is identified by the incorporation of the elongator tRNA to the A-site as a ternary complex with EF-Tu and GTP. The matching of the anticodon of the elongator tRNA and the mRNA codon sequences contributes to the stabilization of the tRNA binding at the A-site and the subsequent hydrolysis of the GTP molecule. The EF-Tu in GDP having less affinity for the ribosome detaches itself from the tRNA and leaves the ribosome. Free in solution, the EF-Tu-GDP interacts with EF-Ts, the elongation factor capable of exchanging the GDP to GTP on EF-Tu, enabling the last mentioned to form additional ternary complexes with elongator tRNAs. At this stage the second moment is recognized by a conformational swing of the tRNA to the peptidyl transferase center of the 50S ribosomal subunit. This accommodation step results in the spontaneous formation of a peptide bond through the interaction between the amino acid of the A-site-bound aa-tRNA and that of the P-site bound peptidyl-tRNA (pept-tRNA). The consequence 31.

(201) of this peptidyl transfer is the addition of one amino acid of the A-site bound tRNA and the deacylation of tRNA in the P site. The last moment of the cycle is known as the translocation. Both the tRNAs and the mRNA are shifted by one codon in a movement facilitated by the elongation EF-G, a process also involving the hydrolysis of its GTP molecule. This last cycle gives a ribosome ready for an additional round of elongation, with an empty A-site for the next cognate (codon specific) elongator tRNA-EF-Tu-GTP ternary complex, an amino acylated tRNA in the P-site and a deacylated tRNA in the E-site. Elongation factor EF-Tu The elongation factor EF-Tu, a member of the super family of guanine nucleotide-binding proteins (GTPases), is encoded by two unlinked genes tufA and tufB located approximately 660 kbp (kilo base pair) apart on the chromosome (140-144). The molecule has three major structural domains (see figure 14), a size of ca. 44 kDa and is the most abundant protein in Escherichia coli with approximately 100.000 molecules per cell, representing about 5 % of the total cellular proteins (145). In the cell, the GTP activated form of elongation factor EF-Tu (EF-Tu•GTP) is responsible for delivering the amino acylated tRNA to the ribosomal A-site by forming a ternary complex tRNA•EF-Tu•GTP, with it (146). The ternary complex was successfully crystallized in 1995 by Nissen et al. (147) and in 1997, a Cryo-EM structure of the ternary complex on the ribosome was obtained (148). With advances in both X-ray and Cryo-EM techniques more novel structures of EF-Tu blocked under various steps of the elongation process are seeing the light (149). The interaction with the amino acylated tRNA has been shown to occur with particular residues in the acceptor helix (the CCA-end and the phosphorylated 5’end) and the domain interfaces and the tRNA T-stem helix with the surface of the E-barrel of domain 3 (147) (See Figure 16). Some of the EF-Tu residues involved in this interaction are universally conserved. As a result of several years of intensive studies, the function of EF-Tu in elongation can be summarized by (i) the initial binding of the ternary complex to the 70S ribosome and the codon-anticodon recognition and (ii) the formation of a stable Ribosome•EF-Tu•aa-tRNA complex. The formation of this stable complex activates the hydrolysis of the GTP molecule to GDP and Pi. The release of the inorganic phosphate Pi in its turn triggers a conformational change of the EF-Tu•GDP molecule. This engenders a reduction of its affinity to the aa-tRNA and subsequently the ribosome from which it dissociates (iii), thus leaving the aa-tRNA bound to A-site (3,74,150). It has been suggested (151) that the ternary complex is brought to the ribosome primarily through an interaction between EF-Tu helix D with helices 4 and 5 of the C-terminal domain (CTD) of one (out of four) L7/L12 protein, in a similar manner to the EF-Tu protein’s interaction with EF-Ts (152,153). 32.

(202) The codon recognition step, which implies the proper orientation of the ternary complex in relation to the bound P-site tRNA for codon and anticodon contact, occurs in the decoding center of the 70S ribosome (154). The formation of a stable cognate codon-anticodon results in the conformational change of the conserved bases of the 16S rRNA (A1492, A 1493 and G530) (21), a possible start process of active site rearrangement (induced fit) for the catalysis of the GTP hydrolysis.. Figure 16. Representation of the structure of elongation factor EF-Tu in complex with guanine nucleotides and tRNA. (A) Crystal structure of Thermus aquaticus EF-Tu in complex with GDP (PDB entry 1TUI). (B) Crystal structure of Thermus aquaticus of EF-G in complex with GTP (PDB entry 1EFT). (C) Crystal structure of the EF-Tu in complex with Phe-tRNA Phe and GTP. The EF-Tu domains are illustrated by roman numbers. The E-sheets are shown in yellow and D-helices in aquamarine. The switch I is in red and the switch II is in cyan whereas the G-nucleotides are in magenta. The aa-tRNA is illustrated in grey and the space filled atoms represent the location of residue Gln97. The pictures were adapted from (155).. The structural mechanism of the hydrolysis of the guanine triphosphate remains unknown to this date. Interestingly enough, it was observed that when a cognate aa-tRNA was bound to the ribosome the structural alterations within the decoding center led to a conformation change of the 30S subunit from an open to closed state, relatively to the 50S, an adjustment not seen for the near-cognate cases (16). It was therefore concluded that the ribosomal changes resulting from the cognate codon-anticodon interaction reconfigured the position of the ternary complex to allow the hydrolysis of the GTP molecule and the subsequent rotation of the aa-tRNA towards the PTC. The GTP hydrolysis on EF-Tu is thought to happen upon the rearrangement of its G-domain. Residues Gly83 and His84 have recently been suggested as favorable mediators of the GTP hydrolysis by respectively positioning a catalytic water molecule and providing the reaction’s ionic condition (151,156,157). The recent Cryo-EM map of the ternary complex PhetRNAPhe•EF-Tu•GDP with kirromycin revealed a kink in the anticodon helix of the aa-tRNA that might correspond to the overall conformational move33.

(203) ment required for the hydrolysis of the GTP molecule(67). This structural adaptation of the tRNA molecule with respect to EF-Tu seems to engender a movement of the stalk base causing the tRNA elbows to interact with the GAC. Close to it is the sarcin-ricin loop (SRL) believed to be in close contact with the GTPase center of EF-Tu. These interactions point towards the transition of the GAC from an open to close form upon ternary complex formation. The hydrolysis of GTP causing the release of the EF-Tu molecule bound to GDP could result in the accommodation of the tRNA in the A-site through a string-like mechanism (158). In addition, a molecular mimicry has been suggested due to the resemblance of the overall shape of the ternary complex with the structure of EF-G in complex with GDP (see Figure 19). In solution, the exchange of GDP to GTP is made possible through the interaction of EF-Tu with the second elongation factor, EF-Ts, a nucleotide exchange factor (GEF). Elongation factor EF-Ts The second elongation factor EF-Ts is the nucleotide exchange factor for the elongation factor EF-Tu (159). EF-Ts binds the EF-Tu•GDP complex in solution mediating the release of the GDP and the subsequent formation of the EF-Ts•EF-Tu•GTP thanks to the high cellular concentration of GTP. The EF-Ts looses its affinity to EF-Tu from which it releases (160). Elongation factor EF-Ts is encoded in Escherichia coli by the single tsf gene and is 282 amino acid long with a total molecular mass of 30.3 kDa (161). The crystal structure of Escherichia coli EF-Tu•EF-Ts complex has revealed, that the EF-Ts is an elongated protein consisted of four domains (See Figure 17). The N-terminal domain has three anti-parallel D-helices (residues 1-54), the core domain (residues 55-179 and 229-263) has two central threestranded anti-parallel E-sheets surrounded by D-helices, the dimerization domain (residues 180-228) has three D-helices (9, 10 and 11) inserted in to the core domain (sub-domain C) and the C-terminal domain has only one Dhelix (162). The core domain which is further divided into sub-domains N (residues 55-140) and C (residues 141-179 and 229-263), has an internal pseudo-symmetry relating the two sheets and some of the helices. The protruding motif, denoted coiled-coil, of sub-domain C (helices 10, 11, residues 187-203 and 208-226) has been shown to be responsible for the dimerization of the EF-Ts in the obtained quaternary [EF-Ts•EF-Tu]2 crystal structure (163,164). From the crystal structure, domain I of EF-Tu is in contact with both EFTs N-terminal domain and sub-domain N of the core domain, whereas the tip of domain 3 of EF-Tu interacts with sub-domain C. Thus domains 1 and 3 of EF-Tu are kept apart in a conformation that is believed to facilitate the dissociation event of the GDP and the following substitution with GTP (165168). 34.

(204) Figure 17. Representation of the structure of the Escherichia coli heterodimer of the elongation factor EF-Ts in complex with elongation factor EF-Tu (Adapted picture from (169)). The ribbon image corresponds to EF-Ts whereas the trace portrayal shows EF-Tu. (A) Position of the EF-Tu domains I (G-domain), II and III with respect to the N-terminal domain (NTD) and core domain of EF-Ts. (B) 90° vertical axis rotation of (A) illustrating the protrusion of the coiled-coil motif (residues 187-226) and the C-terminal module (CTD). The PDB entry is 1EFU.. The process of GDP release from the EF-Ts•EF-Tu complex has been suggested to occur through a conformational change of the guanine recognition loop (the NKXD motif) of EF-Tu by the synchronized outcome of a three step mechanism. (i) Upon binding to EF-Ts the affinity of EF-Tu to the GDP molecule is considerably lowered by the disruption of the Mg2+ ion binding site, which is seen as the intrusion of the side chains of the EF-Ts residues Asp80 and Phe81 into the space between helices B and C of EF-Tu domain 1. (ii) The binding of one E phosphate oxygen is destabilized by the flipping of a peptide in the P-loop (phosphate binding loop) of EF-Tu. (iii) The movement of EF-Tu helix D disturbs the binding of the sugar and base of the GDP (163,170). The structural mechanisms for the binding of GTP to the EF-Ts•EF-Tu complex and the following dissociation of EF-Ts from the EF-Ts•EF-Tu •GTP complex have still not been characterized. Elongation factor EF-G The third and last of the elongation factors is named EF-G. It belongs to the GTPase family of proteins (171). The gene that encodes this protein is known as FusA in Escherichia coli. The protein contains 703 amino acids and has a molecular weight of 77.5 kDa. The binding site of EF-G on the ribosome has been shown to overlap with that of the initiation factor IF2, the elongation factor EF-Tu and both class I release factors RF1 and RF2 (Liljas A. in (172)). The resolved crystal structures of EF-G revealed the protein to 35.

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