Laura Lancaster

Following translation by single ribosomes one codon at a time

We have followed individual ribosomes as they translate single messenger RNA hairpins tethered by the ends to optical tweezers. Here we reveal that translation occurs through successive translocation-and-pause cycles. The distribution of pause lengths, with a median of 2.8 s, indicates that at least two rate-determining processes control each pause. Each translocation step measures three bases—one codon—and occurs in less than 0.1 s. Analysis of the times required for translocation reveals, surprisingly, that there are three substeps in each step. Pause lengths, and thus the overall rate of translation, depend on the secondary structure of the mRNA; the applied force destabilizes secondary structure and decreases pause durations, but does not affect translocation times. Translocation and RNA unwinding are strictly coupled ribosomal functions.

Crystal structure of a translation termination complex formed with release factor RF2

We report the crystal structure of a translation termination complex formed by the Thermus thermophilus 70S ribosome bound with release factor RF2, in response to a UAA stop codon, solved at 3 Å resolution. The backbone of helix α5 and the side chain of serine of the conserved SPF motif of RF2 recognize U1 and A2 of the stop codon, respectively. A3 is unstacked from the first 2 bases, contacting Thr-216 and Val-203 of RF2 and stacking on G530 of 16S rRNA. The structure of the RF2 complex supports our previous proposal that conformational changes in the ribosome in response to recognition of the stop codon stabilize rearrangement of the switch loop of the release factor, resulting in docking of the universally conserved GGQ motif in the PTC of the 50S subunit. As seen for the RF1 complex, the main-chain amide nitrogen of glutamine in the GGQ motif is positioned to contribute directly to catalysis of peptidyl-tRNA hydrolysis, consistent with mutational studies, which show that most side-chain substitutions of the conserved glutamine have little effect. We show that when the H-bonding capability of the main-chain N-H of the conserved glutamine is eliminated by substitution with proline, peptidyl-tRNA esterase activity is abolished, consistent with its proposed role in catalysis.

The ribosome uses two active mechanisms to unwind messenger RNA during translation.

The ribosome translates the genetic information encoded in messenger RNA into protein. Folded structures in the coding region of an mRNA represent a kinetic barrier that lowers the peptide elongation rate, as the ribosome must disrupt structures it encounters in the mRNA at its entry site to allow translocation to the next codon. Such structures are exploited by the cell to create diverse strategies for translation regulation, such as programmed frameshifting, the modulation of protein expression levels, ribosome localization and co-translational protein folding. Although strand separation activity is inherent to the ribosome, requiring no exogenous helicases, its mechanism is still unknown. Here, using a single-molecule optical tweezers assay on mRNA hairpins, we find that the translation rate of identical codons at the decoding centre is greatly influenced by the GC content of folded structures at the mRNA entry site. Furthermore, force applied to the ends of the hairpin to favour its unfolding significantly speeds translation. Quantitative analysis of the force dependence of its helicase activity reveals that the ribosome, unlike previously studied helicases, uses two distinct active mechanisms to unwind mRNA structure: it destabilizes the helical junction at the mRNA entry site by biasing its thermal fluctuations towards the open state, increasing the probability of the ribosome translocating unhindered; and it mechanically pulls apart the mRNA single strands of the closed junction during the conformational changes that accompany ribosome translocation. The second of these mechanisms ensures a minimal basal rate of translation in the cell; specialized, mechanically stable structures are required to stall the ribosome temporarily. Our results establish a quantitative mechanical basis for understanding the mechanism of regulation of the elongation rate of translation by structured mRNAs.

Crystal structures of EF-G-ribosome complexes trapped in intermediate states of translocation.

Introduction: One of the most critical and complex steps of protein synthesis is the coupled translocation of mRNA and tRNAs (mRNA and tRNAs) through the ribosome, catalyzed by the guanosine triphosphatase (GTPase) elongation factor EF-G. Although several of the main steps have been identified, the underlying molecular mechanisms of translocation are poorly understood. A central question is how structural rearrangements in the ribosome are coupled to movement of mRNA and tRNA. Methods: We trapped and crystallized complexes of Thermus thermophilus ribosomes bound with EF-G, mRNA, and tRNA, using the antibiotic fusidic acid (which prevents release of EF-G after GTP hydrolysis) or the nonhydrolyzable GTP analog GDPNP, in intermediate states of translocation. Their crystal structures were determined to resolutions from 3.5 to 4.1 Å. Results: The structures of the fusidic acid complex (Fus) and two GDPNP complexes (GDPNP-I and GDPNP-II) reveal conformational changes occurring during intermediate states of translocation, including large-scale (15° to 18°) rotation of the 30S subunit head and 3° to 5° rotation of the 30S body. In all complexes, the tRNA acceptor end has moved from the 50S subunit P site to the 50S E site, while the anticodon stem loop (ASL) and mRNA move with the head of the 30S subunit to positions between the P and E sites, forming chimeric pe*/E intermediate states. The elongated, mobile domain IV of EF-G moves to contact the head of the 30S subunit and the backbone of the mRNA. Two universally conserved bases of 16S rRNA that intercalate between bases of the mRNA may act as “pawls” of a translocational ratchet. In the GDPNP complexes, structuring of the conserved switch loop I segment, which was disordered in previous structures, completes the cage that encloses GDPNP and fixes the relative geometry of EF-G domains I, III, and V. In the Fus complex, the position of fusidic acid overlaps that of switch loop I, stabilizing contacts between domains I and III that are normally made by the structured switch loop. Conclusion: Our structures capture intermediate states of the rate-limiting step of translocation, in which movement of the tRNA ASL and mRNA is coupled to rotational movement of the 30S subunit head. Slippage of the translational reading frame during reverse rotation of the head during translocation may be prevented by intercalation of bases C1397 and A1503 of 16S rRNA, which project from the body of the 30S subunit, between mRNA bases. The antibiotic fusidic acid appears to stabilize binding of EF-G to the ribosome in the GDP state by mimicking the structure of the conserved core of switch loop I of EF-G in the GTP state. Revealed in Translation The ribosome, with the help of transfer RNAs (tRNAs), converts the triple genetic code in messenger RNA (mRNA) into protein. Upon decoding of a codon, the mRNA and associated tRNAs must be moved through the ribosome, so that the next codon can be read, with a new charged tRNA taken in at the A (aminoacyl-tRNA) site, the newly extended peptidyl-tRNA moved into the P (peptidyl-tRNA) site, and the deacylated tRNA removed from the exit site in the ribosome (see the Perspective by Rodnina). Crystal structures from Tourigny et al. (p. 1235490), Pulk and Cate (p. 1235970), and Zhou et al. (p. 1236086), variously capture the prokaryotic ribosome during this translocation phase, revealing the hybrid states of the tRNAs and the substantial motions of the 30S ribosomal subunit during the process, the role of elongation factor G, and suggest how the direction and reading frame of the mRNA is maintained. Crystal structures reveal how messenger RNA and transfer RNAs transition through the prokaryotic ribosome during translation. [Also see Perspective by Rodnina] Translocation of messenger and transfer RNA (mRNA and tRNA) through the ribosome is a crucial step in protein synthesis, whose mechanism is not yet understood. The crystal structures of three Thermus ribosome-tRNA-mRNA–EF-G complexes trapped with β,γ-imidoguanosine 5′-triphosphate (GDPNP) or fusidic acid reveal conformational changes occurring during intermediate states of translocation, including large-scale rotation of the 30S subunit head and body. In all complexes, the tRNA acceptor ends occupy the 50S subunit E site, while their anticodon stem loops move with the head of the 30S subunit to positions between the P and E sites, forming chimeric intermediate states. Two universally conserved bases of 16S ribosomal RNA that intercalate between bases of the mRNA may act as “pawls” of a translocational ratchet. These findings provide new insights into the molecular mechanism of ribosomal translocation.

Orientation of ribosome recycling factor in the ribosome from directed hydroxyl radical probing.

Ribosome recycling factor (RRF) disassembles posttermination complexes in conjunction with elongation factor EF-G, liberating ribosomes for further rounds of translation. The striking resemblance of its L-shaped structure to that of tRNA has suggested that the mode of action of RRF may be based on mimicry of tRNA. Directed hydroxyl radical probing of 16S and 23S rRNA from Fe(II) tethered to ten positions on the surface of E. coli RRF constrains it to a well-defined location in the subunit interface cavity. Surprisingly, the orientation of RRF in the ribosome differs markedly from any of those previously observed for tRNA, suggesting that structural mimicry does not necessarily reflect functional mimicry.

Crystal structure of release factor RF3 trapped in the GTP state on a rotated conformation of the ribosome.

The class II release factor RF3 is a GTPase related to elongation factor EF-G, which catalyzes release of class I release factors RF1 and RF2 from the ribosome after termination of protein synthesis. The 3.3 Å crystal structure of the RF3·GDPNP·ribosome complex provides a high-resolution description of interactions and structural rearrangements that occur when binding of this translational GTPase induces large-scale rotational movements in the ribosome. RF3 induces a 7° rotation of the body and 14° rotation of the head of the 30S ribosomal subunit, and itself undergoes inter- and intradomain conformational rearrangements. We suggest that ordering of critical elements of switch loop I and the P loop, which help to form the GTPase catalytic site, are caused by interactions between the G domain of RF3 and the sarcin-ricin loop of 23S rRNA. The rotational movements in the ribosome induced by RF3, and its distinctly different binding orientation to the sarcin-ricin loop of 23S rRNA, raise interesting implications for the mechanism of action of EF-G in translocation.

How the ribosome hands the A-site tRNA to the P site during EF-G–catalyzed translocation

Caught in the act of making protein The ribosome is a large RNA-protein complex that converts the genetic code stored in messenger RNA (mRNA) into proteins. Zhou et al. have determined the structure of a bacterial ribosome caught in the act of decoding an mRNA. Transfer RNAs (tRNAs) decipher the genetic code in the mRNA to ensure that the ribosome uses the correct amino acids. The structure shows tRNAs in the process of being moved between successive protein-building binding pockets as the ribosome reads the mRNA like a piece of old-fashion computer tape. Science, this issue p. 1188 The structure of an intermediate shows how the ribosome moves transfer RNAs from one binding site to another. Coupled translocation of messenger RNA and transfer RNA (tRNA) through the ribosome, a process catalyzed by elongation factor EF-G, is a crucial step in protein synthesis. The crystal structure of a bacterial translocation complex describes the binding states of two tRNAs trapped in mid-translocation. The deacylated P-site tRNA has moved into a partly translocated pe/E chimeric hybrid state. The anticodon stem-loop of the A-site tRNA is captured in transition toward the 30S P site, while its 3′ acceptor end contacts both the A and P loops of the 50S subunit, forming an ap/ap chimeric hybrid state. The structure shows how features of ribosomal RNA rearrange to hand off the A-site tRNA to the P site, revealing an active role for ribosomal RNA in the translocation process.

Visualization of two transfer RNAs trapped in transit during elongation factor G-mediated translocation

Significance One of the most critical and complex steps of the protein synthesis elongation cycle is the coupled translocation of messenger (m)RNA and the A- and P-site transfer (t)RNAs through the ribosome, catalyzed by the elongation factor EF-G. This step involves large-scale molecular movements in the ribosome, including rotational movements of the body and head of the 30S subunit. Previously, structures have been obtained for trapped intermediates containing a single tRNA. Here, we report the cryo-EM structure of an intermediate trapped with both tRNAs. This structure represents a previously missing link in understanding the mechanism of translocation, revealing that the ribosome uses two distinct molecular ratchets, involving both intra- and intersubunit rotational movements, to drive the synchronous movement of tRNAs and mRNA. During protein synthesis, coupled translocation of messenger RNAs (mRNA) and transfer RNAs (tRNA) through the ribosome takes place following formation of each peptide bond. The reaction is facilitated by large-scale conformational changes within the ribosomal complex and catalyzed by elongtion factor G (EF-G). Previous structural analysis of the interaction of EF-G with the ribosome used either model complexes containing no tRNA or only a single tRNA, or complexes where EF-G was directly bound to ribosomes in the posttranslocational state. Here, we present a multiparticle cryo-EM reconstruction of a translocation intermediate containing two tRNAs trapped in transit, bound in chimeric intrasubunit ap/P and pe/E hybrid states. The downstream ap/P-tRNA is contacted by domain IV of EF-G and P-site elements within the 30S subunit body, whereas the upstream pe/E-tRNA maintains tight interactions with P-site elements of the swiveled 30S head. Remarkably, a tight compaction of the tRNA pair can be seen in this state. The translocational intermediate presented here represents a previously missing link in understanding the mechanism of translocation, revealing that the ribosome uses two distinct molecular ratchets, involving both intra- and intersubunit rotational movements, to drive the synchronous movement of tRNAs and mRNA.

Interactions and dynamics of the Shine Dalgarno helix in the 70S ribosome.

The crystal structure of an initiation-like 70S ribosome complex containing an 8-bp Shine–Dalgarno (SD) helix was determined at 3.8-Å resolution. Translation–libration–screw analysis showed that the inherent anisotropic motions of the SD helix were biased along its helical axis, suggesting that during the first step of translocation, the SD helix moves along its helical screw axis. Contacts between the SD helix and the ribosome were primarily through interactions with helices 23a, 26, and 28 of 16S rRNA. Contact with the neck (helix 28) of the 30S subunit near its hinge point suggests that formation of the SD helix could affect positioning of the head of the 30S subunit for optimal interaction with initiator tRNA. The bulged U723 in helix 23a interacts with the minor groove of the SD helix at the C1539·G-10 base pair, explaining its selective conservation in bacteria and archaea.

Ribosomal protein S1 unwinds double-stranded RNA in multiple steps

The sequence and secondary structure of the 5′-end of mRNAs regulate translation by controlling ribosome initiation on the mRNA. Ribosomal protein S1 is crucial for ribosome initiation on many natural mRNAs, particularly for those with structured 5′-ends, or with no or weak Shine-Dalgarno sequences. Besides a critical role in translation, S1 has been implicated in several other cellular processes, such as transcription recycling, and the rescuing of stalled ribosomes by tmRNA. The mechanisms of S1 functions are still elusive but have been widely considered to be linked to the affinity of S1 for single-stranded RNA and its corresponding destabilization of mRNA secondary structures. Here, using optical tweezers techniques, we demonstrate that S1 promotes RNA unwinding by binding to the single-stranded RNA formed transiently during the thermal breathing of the RNA base pairs and that S1 dissociation results in RNA rezipping. We measured the dependence of the RNA unwinding and rezipping rates on S1 concentration, and the force applied to the ends of the RNA. We found that each S1 binds 10 nucleotides of RNA in a multistep fashion implying that S1 can facilitate ribosome initiation on structured mRNA by first binding to the single strand next to an RNA duplex structure (“stand-by site”) before subsequent binding leads to RNA unwinding. Unwinding by multiple small substeps is much less rate limited by thermal breathing than unwinding in a single step. Thus, a multistep scheme greatly expedites S1 unwinding of an RNA structure compared to a single-step mode.