Translation in Prokaryotes

Abstract

This review summarizes our current understanding of translation in prokaryotes, focusing on the mechanistic and structural aspects of each phase of translation: initiation, elongation, termination, and ribosome recycling. The assembly of the initiation complex provides multiple checkpoints for messenger RNA (mRNA) and start-site selection. Correct codon-anticodon interaction during the decoding phase of elongation results in major conformational changes of the small ribosomal subunit and shapes the reaction pathway of guanosine triphosphate (GTP) hydrolysis. The ribosome orchestrates proton transfer during peptide bond formation, but requires the help of elongation factor P (EF-P) when two or more consecutive Pro residues are to be incorporated. Understanding the choreography of transfer RNA (tRNA) and mRNA movements during translocation helps to place the available structures of translocation intermediates onto the time axis of the reaction pathway. The nascent protein begins to fold cotranslationally, in the constrained space of the polypeptide exit tunnel of the ribosome. When a stop codon is reached at the end of the coding sequence, the ribosome, assisted by termination factors, hydrolyzes the ester bond of the peptidyl-tRNA, thereby releasing the nascent protein. Following termination, the ribosome is dissociated into subunits and recycled into another round of initiation. At each step of translation, the ribosome undergoes dynamic fluctuations between different conformation states. The aim of this article is to show the link between ribosome structure, dynamics, and function.

Figure 1.

Kinetic model of translation initiation. (Top) Assembly of the 30S preinitiation complex (PIC) and 30S initiation complex (IC). Arrival times are calculated using experimentally measured bimolecular association rate constants and the in vivo concentrations of initiation factors in E. coli. Residence times are calculated from the measured dissociation rate constants of the individual components; mRNA binding is shown as a last step, but can occur at any step of the assembly pathway, independent of the presence of initiation factors or fMet-tRNA^fMet. Recognition of the start codon signifies the transition to the 30S IC (based on data in Milon et al. 2012). (Middle) Formation and maturation of the 70S IC. After subunit joining, IF3 may remain loosely bound to a site on the large subunit (LSU) (based on data in Goyal et al. 2017). (Bottom) Checkpoints of mRNA selection. From an mRNA-centric point of view, structured mRNAs can be recruited to the platform of the small subunit (SSU), unfold, and then accommodate in the mRNA-binding channel of the SSU (based on data in Milon et al. 2008 and Milon and Rodnina 2012).

Figure 2.

Structural mechanism of decoding as visualized by cryoelectron microscopy (cryo-EM). (Top) Intermediates of cognate decoding by elongation factor (EF)-Tu•GDP–Phe-tRNA^Phe. (Left) Schematic of the cognate codon–anticodon interaction between the UUC mRNA codon and the AAG anticodon of Phe-tRNA^Phe. Other panels show decoding intermediates from the T state prior to codon reading, A*/T, where the codon has been recognized but EF-Tu did not move onto the sarcin-ricin loop (SRL) of the SSU and A/T state with the correct codon–anticodon interaction and EF-Tu docked on the SRL. Insets on top show the orientation of the G-domain of EF-Tu relative to the SRL. GCP, nonhydrolyzable GTP analog GDPCP. Insets at the bottom show the codon–anticodon complex and the key residues of 16S ribosomal RNA (rRNA) interacting with it. (Middle) Same as above for a near-cognate pair with a single G–U mismatch in the second position of the codon–anticodon complex. (Bottom) Intermediates of cognate decoding by SelB•GDPNP/Sec-tRNA^Sec. GNP, nonhydrolyzable GTP analog GDPNP; IB, initial binding prior to codon reading; CR, codon reading complex in which the anticodon of the tRNA comes into the proximity of the codon, but prior to base pairing; GA, GTPase-activated complex analogous to the A/T state. (Figure was prepared using structure coordinates from Fischer et al. 2016 and Loveland et al. 2017, PDB 5UYK, 5UYL, 5UYM, 5UYN, 5UYP, 5UYQ, 5LZB, 5LZC and 5LZD.)

Figure 3.

Models for proton transfer during peptide bond formation. Reaction schemes are shown for the eight-membered proton shuttle and proton wire mechanisms. P-site tRNA is shown in green, A-site tRNA in blue, and 2′OH of A2451 in black. The nucleophilic attack is depicted by red arrows. In the eight-membered proton shuttle, the attack of the α-amino group on the ester carbonyl carbon results in an eight-membered rate-limiting transition state in which a proton from the α-amino group is received by the 2′OH group of A76, which at the same time donates its proton to the carbonyl oxygen via an adjacent water molecule (Kuhlenkoetter et al. 2011). In the proton wire model, the proton from the α-amino group is received by the 2′OH group of A76, which in turn donates a proton to the 2′OH of A2451, and then to a water molecule (W1), which is partially negatively charged (Polikanov et al. 2014). Both models account for the concerted movement of three protons in the rate-limiting transition state (Kuhlenkoetter et al. 2011). (From Polikanov et al. 2014; adapted, with permission, from Nature Publishing Group under a Creative Commons license.)

Figure 4.

Action of elongation factor P (EF-P) on ribosomes stalled at polyproline stretches. (A) Ribosomes stall during translation of proteins containing three consecutive prolines (red stars). Binding of the peptidyl-tRNA (green) to the P site is destabilized, which (B) can lead to peptidyl-tRNA drop-off. (C) The all-trans or all-cis conformations of polyprolines of the nascent chain are not possible because of a steric clash with G2061 (gray) within the tunnel wall. Peptidyl-tRNA is destabilized and prevents accommodation of the A-site tRNA (orange) and peptide bond formation. (D) Ribosomes stalled on polyproline stretches are recognized by EF-P (pink), which binds within the E-site region and stabilizes the peptidyl-tRNA. EF-P binding is facilitated via contacts with the L1 stalk and the P-site tRNA as well as E-site codon. (E) Interaction of the ε(R)-β-lysyl-hydroxylysine with the CCA-end of P-site tRNA^Pro stabilizes the P-site tRNA as well as the nascent chain, by forcing the prolines to adopt an alternative conformation that passes into the ribosomal exit tunnel. (F) Thus, an optimal geometry between the nascent chain and the aminoacyl-tRNA in the A site is achieved and peptide bond formation can occur. (From Huter et al. 2017; adapted, with permission, from Elsevier © 2017.)

Figure 5.

Kinetic model of translocation. The rotation states of the small subunit (SSU) relative to the large subunit (LSU) (gray) are indicated by color intensity of the SSU body (light blue for nonrotated, N; dark blue for rotated, R). The swiveling motions of the SSU head are depicted by a color gradient from light green (classical nonswiveled SSU head position) to forest green (maximum degree of SSU head domain swiveling relative to the SSU body domain) (Belardinelli et al. 2016a). Peptidyl- and deacylated tRNA in the pretranslocation state (PRE) complex are shown in magenta and blue, respectively. EF-G (purple) is shown in two conformations, a compact (Lin et al. 2015) and an extended one after engagement with the ribosome (Ramrath et al. 2013; Zhou et al. 2014). The rates of transitions between PRE(N) and PRE(R) and PRE(N)–EF-G and PRE(R)–EF-G (k_CW and k_CCW at 37°C) are modified from data in Sharma et al. (2016b). The rate constants for the kinetically defined steps 1, 2, 3, 4, and 5 are from ensemble kinetics studies at 37°C (Belardinelli et al. 2016a). Step 1, EF-G binding. Step 2, GTP hydrolysis (Rodnina et al. 1997; Savelsbergh et al. 2003) and opening of the SSU because of the opposite movements of the SSU head and body domains (see inset) (Belardinelli et al. 2016a). Step 3, unlocking of the tRNA–mRNA complex on the SSU, which is rate-limiting for tRNA movement and Pi release from EF-G (Savelsbergh et al. 2003). The existence of rapid transitions between steps 3 and 4 was shown using stalled translocation intermediates (Savelsbergh et al. 2003; Holtkamp et al. 2014). Translocation intermediates (CHI1 to CHI4) are adopted from smFRET data (Adio et al. 2015). The posttranslocation (POST) state may entail further conformational substates (Wasserman et al. 2016). The light red background indicates complexes undergoing unlocking; the light green background shows complexes that move toward relocking. (Inset) Distinct timing of counterclockwise (CCW) and clockwise (CW) movements of the SSU body relative to LSU (blue symbols) and of the SSU head (green symbols) (Belardinelli et al. 2016a). (From Belardinelli et al. 2016b; with permission from Taylor & Francis and Creative Commons Public Domain licensing.)

Figure 6.

Cotranslational protein folding. Callouts summarize the potential effects at each step (Rodnina 2016). U, unfolded state; C, compact transient state or folding intermediate; N, native fold.