key: cord-0982270-naeg0din
authors: Bao, Chen; Ermolenko, Dmitri N.
title: Ribosome as a Translocase and Helicase
date: 2021-07-21
journal: Biochemistry (Mosc)
DOI: 10.1134/s0006297921080095
sha: 9c5159088dfad01d8cf1f919d9c36721d214b388
doc_id: 982270
cord_uid: naeg0din

During protein synthesis, ribosome moves along mRNA to decode one codon after the other. Ribosome translocation is induced by a universally conserved protein, elongation factor G (EF-G) in bacteria and elongation factor 2 (EF-2) in eukaryotes. EF-G-induced translocation results in unwinding of the intramolecular secondary structures of mRNA by three base pairs at a time that renders the translating ribosome a processive helicase. Professor Alexander Sergeevich Spirin has made numerous seminal contributions to understanding the molecular mechanism of translocation. Here, we review Spirin’s insights into the ribosomal translocation and recent advances in the field that stemmed from Spirin’s pioneering work. We also discuss key remaining challenges in studies of translocase and helicase activities of the ribosome.

During the elongation phase of protein synthesis, the ribosome decodes sequences of codons by binding of the tRNA molecules charged with amino acids. Both small (30S in bacteria) and large (50S in bacteria) subunits con tain three tRNA binding sites: the A (aminoacyl), the P (peptidyl) and the E (exit) sites. At the beginning of elon gation cycle, the newly arrived aminoacyl tRNA binds to the A site of the ribosome. Following the peptidyl transfer reaction, the resulting peptidyl and deacylated tRNAs together with associated mRNA codons are translocated from the A and P to P and E sites, respectively ( Fig. 1, a f) . The process is catalyzed by a universally conserved pro tein factor EF G (EF 2 in eukaryotes). The molecular mechanism of translocation has fascinated scientists since the inception of the proteins synthesis field and remained one of the main areas of research in the labora tory of Alexander Spirin for over 50 years [1 6]. Spirin's laboratory has discovered a number of important facets of the translocation mechanism [7 11]. Spirin's lockingunlocking [2, 6] and Brownian ratchet [4, 12, 13] models of ribosomal translocation were highly influential and provided framework for investigations of translocation for decades. Below we review key contributions of the Alexander Spirin's laboratory and recent progress in investigations of ribosomal translocation that stemmed from the Spirin's ideas. More comprehensive reviews of the current state of understanding of the translocation mechanism can be found elsewhere [14 16 ].

Relative to the uncatalyzed reaction, the universally conserved GTPase, EF G, accelerates the rate of ribo some translocation by ∼four orders of magnitude [18 20] . GTP hydrolysis by EF G is activated by the interaction of the G domain of this protein with the sarcin-ricin loop (SRL) of the 23S rRNA [21, 22] . Works of Kaziro's and Spirin's laboratories established the role of GTP hydrolysis in translocation [9, 23 25] . They demonstrat ed that EF G induces efficient translocation in the GTP bound form. In the presence of GDP or in the absence of nucleotides, EF G does not promote translocation. Replacing GTP with non hydrolysable analogues pre serves the ability of EF G to induce translocation that was measured by the increase in puromycin reactivity of peptidyl tRNA or by the release of deacylated tRNA from the ribosome. However, the non hydrolysable ana BIOCHEMISTRY (Moscow) logues of GTP trap EF G on the ribosome. These results suggest that GTP hydrolysis is not required for transloca tion but it is essential for EF G release. Consistent with the Spirin-Kaziro experiments, more recent kinetic studies have shown that the non hydrolysable analogues of GTP do not alter the translocation pathway [26, 27] . Furthermore, replacing GTP with non hydrolysable ana logues only moderately affects the rate of a single round of translocation, reducing it by 2 50 folds depending on experimental conditions [19, 26, 28, 29] .

Another fundamental aspect of the translocation mechanism was discovered by Spirin and his colleagues in the experiments demonstrating that tRNAs can translo cate through the ribosome in the absence of mRNA [10, 11] . These observations suggest that the movement of mRNA is driven by the translocation of the associated anti codon stem loops (ASLs) of A and P site tRNAs. More recent studies indicate that mRNA translocation requires the presence of ASL in the A site and full length tRNA in the P site of the pre translocation ribosome [30] . Consistent with the idea of tRNA driven translocation of mRNA, single molecule measurements showed that mRNA translocates three nucleotides at a time without detectable sub steps [31] . Since tRNAs interact with both 30S and 50S subunits (Fig. 1, a f) , the tRNA driven mechanism of translocation indicates that both ribosomal subunits are involved in this process. and deacylated (orange) tRNAs relative to the A (cyan), P (grey), and E (yellow) sites on the 50S subunit and 30S head and body. d f) Structural view from the subunit interface of the 70S ribosomes bound with tRNAs and EF G, in which 50S and 30S are shown in blue and yellow with transparency, tRNAs in solid orange and green, EF G in solid red. g i) Intersubunit rotation accompany ing translocation viewed from the solvent side of the 30S. The 50S and 30S are displayed in blue and yellow, and the counter clockwise rota tion of 30S relative to 50S is indicated by the arrows. Degrees of intersubunit rotation (from [17]) measured relative to the structure of non rotated ribosome (PDBID 4V51). a, d, g) The rotated (R) pre translocation ribosome is bound with EF G and tRNAs in A/P and P/E hybrid states (PDBID 4V7D). b, e, h) The partially rotated ribosome containing tRNAs in ap/P and pe/E tRNAs chimeric (CH) states (PDBID 4W29). c, f, i) The post translocation EF G bound ribosome in nonrotated (NR) conformation (PDBID 4V5F), which contains tRNAs bound in classical P/P and E/E state. ed that (i) tRNA translocation involves transition from the "locked" to the "unlocked" ribosome conformation that facilitates tRNA diffusion through the ribosome; (ii) peptidyl transferase reaction triggers formation of an intermediate of translocation in which tRNAs are shifted on the large subunit but not yet translocated on the small subunit; (iii) translocation involves movement of the ribo somal subunits relative to each other. Similar ideas were independently proposed by M. S. Bretscher [32] . As dis cussed below, many aspects of this model turned out to be prophetic. To this day, the rate limiting step of transloca tion is often referred to as "unlocking".

Neutron scattering experiments performed by Spirin, Serdyuk, and May provided an early indirect evi dence for intersubunit rearrangements accompanying translocation [33, 34] . Nevertheless, further verification of the key predictions of the locking-unlocking model took several decades and required developing new exper imental approaches such as chemical probing of RNA structure, cryogenic electron microscopy (cryo EM), and single molecule Förster resonance energy transfer (smFRET) microscopy [35] .

Twenty years after the introduction of the locking-unlocking model, Danesh Moazed and Harry Noller used chemical probing for mapping of the tRNA binding sites to demonstrate that the reaction of transpeptidation triggers spontaneous translocation of the acceptors stems of the resulting peptidyl and deacylated tRNAs from the A and P to P and E sites of the large sub unit, respectively, while tRNA ASLs remain in the origi nal A and P sites of the small subunit [36] . Hence, inter mediate A/P and P/E hybrid states of tRNA binding were formed (Fig. 1, a and d) . Completion of translocation of tRNAs on the small subunit was shown to require EF G and GTP (Fig. 1, a f) .

A decade later, another key prediction of the lock ing-unlocking model was corroborated by cryo EM reconstruction of the EF G-ribosome complex per formed by Joachim Frank and Rajendra Agrawal [37] . These experiments demonstrated that EF G binding induces rotation of the small 30S subunit relative to the large 50S subunit parallel to the plane of the intersubunit interface ( were followed by numerous structural and single mole cule studies that provided unprecedented insights into the structural rearrangements of the ribosome, tRNAs, and EF G accompanying translocation [38, 39] .

Cryo EM and FRET experiments revealed that 6 10° intersubunit rotation is coupled with the movement of peptidyl and deacylated tRNAs into A/P and P/E hybrid states (Fig. 2, a and b) [38, 40 43] . These data established equivalence of the nonrotated and rotated conformations with classical and hybrid states of tRNA binding, respec tively. Cryo EM and smFRET studies have also shown that formation of the rotated, hybrid state of the ribosome is accompanied by the inward movement of the mobile domain of the large ribosomal subunit named L1 stalk (Fig. 2c) , which comprises ribosomal protein uL1 and helices 76, 77, and 78 of the 23S rRNA [35, 39] . Upon transition from the open to closed conformation, the extremity of the L1 stalk moves by as much as 60 Å and the L1 stalk becomes bound to the elbow of the P/E tRNA.

smFRET studies also demonstrated that in the absence of EF G, the pretranslocation ribosome sponta neously fluctuates between the nonrotated, classical and the rotated, hybrid state conformations (Fig. 2, a and b ) [38, 44 46] . Binding of EF G•GTP transiently stabilizes the rotated, hybrid state conformation ( Fig. 1, a, d, and g) ; translocation of mRNA and tRNA on the small subunit is coupled with the reverse transition into non rotated, clas sical state conformation (Fig. 1 , c, f, and i) [26, 40, 47] .

In addition to intersubunit rearrangements and movement of the L1 stalk, translocation is accompanied by large structural changes within the small ribosomal subunit. The small subunit comprises three structural domains: head, body, and platform. Structural studies show that the 30S head rotates by up to 20° relative to the rest of the small subunit around the axis that is orthogo nal to the axis of intersubunit rotation (Fig. 2 , d and e) [48, 49] . In the EF G bound intermediate of transloca tion visualized by X ray crystallography and cryo EM, in which the 30S head is observed in a swiveled conforma tion, two tRNAs are translocated along the 50S subunit and the 30S platform/body but not yet translocated rela tive to the 30S head ( Fig. 1, b , e, and h) [50, 51] . In these positions named ap/P and pe/E chimeric states, tRNAs are trapped midway between hybrid (A/P and P/E) and posttranslocation classical (P/P and E/E states) and like ly represent a late intermediate of translocation.

The tip of domain IV of EF G plays a critical role in translocation activity of EF G and reading frame mainte nance [19, 52 54] . When EF G is bound in the rotated hybrid state conformation of the pretranslocation ribo some, domain IV of EF G is positioned next to ASL of A/P tRNA (Fig. 1, a and d) [55] . Upon translocation, domain IV of EF G docks into the A site of the small sub unit vacated by the peptidyl tRNA (Fig. 1 , c and f) [38, 56] . Hence, upon reverse intersubunit rotation and 30S back swivel, domain IV of EF G displaces ASL of peptidyl tRNA and prevents its backward movement.

Which of the aforementioned conformational rearrangements is the rate limiting step that "unlocks" the ribosome (using Spirin's terminology) and facilitates tRNA translocation is not entirely clear. Reaction of transpeptidation "unlocks" the ribosome in a sense that it enables spontaneous intersubunit rotation and fluctua tions of tRNAs between the classical and hybrid states [44 46] . However, in the absence of EF G, these fluctuations are unproductive and do not lead to tRNA/mRNA translocation on the small subunit [44 46] . Several lines of evidence suggest that the domain IV of EF G destabilizes interactions of A site tRNA with 16S rRNA [57 61 ]. Hence, the EF G induced changes in the A site may "unlock" the ribosome. Finally, anoth er possible "unlocking" rearrangement is swiveling of the 30S head, which opens the path for tRNA movement from P to E site that is otherwise constricted [49] . Further studies are needed to establish complete sequence of the structural rearrangements accompanying translocation and identify the rate limiting step in this process.

Although translation is greatly accelerated by EF G, the Spirin and Pestka laboratories demonstrated that translocation can occur spontaneously in the absence of protein factors [7, 8, 62] . It was found in the Spirin labo ratory that spontaneous translocation is stimulated by modification of the universally conserved ribosomal pro tein (u)S12 of the 30S subunit by thiol specific reagents, which were added to inactivate EF G and thus rule out the presence of trace amounts of this protein [7, 63, 64] . More recent studies indicated that removal of the 30S proteins uS12 and uS13 enhanced the factor free translo cation possibly by weakening tRNA interactions with the 30S A and P sites, respectively [65] . Furthermore, it was reported that a single round factor free translocation could be induced by antibiotics sparsomycin, lincomycin, and chloramphenicol that bind to the 50S A site and thus destabilize the A site tRNA binding [20, 66] .

Based on the observations of factor free transloca tion, Spirin postulated that translocation is an inherent property of the ribosome and that energy of the peptidyl transfer reactions is sufficient to promote tRNA move ment [3] . However, the slow rate of spontaneous translo cation and the observation of reverse spontaneous translocation in some tRNA/mRNA contexts suggest that the reaction of transpeptidation is not the only ener gy source of translocation, which is also promoted by the energy stored in EF G•GTP. Indeed, it was estimated that transpeptidation driven translocation would require ∼80% efficiency of the conversion of chemical energy into mechanical motion [67] . Such high efficiency is untypical for macromolecular motors [67] .

Two alternative idealized models, the power stroke and the Brownian ratchet models, are employed to describe conversion of chemical energy into mechanical work by macromolecular motors [68, 69] . Chemical ener gy may be converted into elastic energy or conformation al transition that drives large conformational change of the macromolecule, i.e., the power stroke. Alternatively, energy of chemical reaction may be used to bias random, thermally driven motions of the macromolecule into uni directional movement. Thus, chemical reaction plays a role that is similar to a pawl directing the movement of a wheel of mechanical ratchet. The chemical change either strictly precedes conformational change (the power stroke) or follows it (the Brownian ratchet) [68] . These two mechanisms can be distinguished by examining the load dependence of the molecular motor movement [68] .

Several groups hypothesized that tRNA transloca tion is mediated by the power stroke of domain IV of EF G driven by GTP hydrolysis [19, 70, 71] . This hypothesis is supported by kinetic data suggesting that GTP hydrol ysis by EF G precedes translocation [19] . However, the Spirin-Kaziro experiments and more recent kinetic measurements show that translocation occurs rapidly and efficiently in the absence of GTP hydrolysis, when GTP is replaced with non hydrolysable analogues [9, 19, 23 26, 28, 29] . These data suggest that GTP hydrolysis by EF G is not coupled with translocation and argue against the GTP hydrolysis driven power stroke.

Based on the observations of spontaneous, factor free translocation, Spirin reasoned that Brownian motions of tRNA are sufficient to explain translocation without invoking the power stroke by EF G [3, 12, 13]. This hypothesis was further corroborated by the smFRET data demonstrating spontaneous intersubunit rotation and fluctuations of tRNA between the classical and hybrid states [44 46 ]. Spirin's ideas were ultimately rein forced by the single molecule optical tweezer measure ments of ribosome translocation against applied force showing that EF G catalyzed translocation is also best described by the Brownian ratchet model [67] .

In the Brownian ratchet mechanism of EF G cat alyzed translocation, EF G•GTP likely acts as a pawl of the ratchet that biases tRNA diffusion through the ribo some and couples translocation with the ribosome dynam ics [14, 15] . The structure of EF G trapped in the rotated pretranslocation ribosome (Fig. 1, a and d) reveals the basis for coupling of intersubunit rotation and tRNA/mRNA translocation [55] . Unproductive spontaneous fluctuations of the ribosome from the rotated into nonrotated confor mation leads to the return of peptidyl tRNA from the hybrid A/P into the classical A/A state. However, when EF G is bound to the rotated pretranslocation ribosome, domain IV of EF G creates steric hindrance for the return of peptidyl tRNA from the hybrid A/P into classical A/A state [55] . Similarly, domain IV of EF G sterically blocks the return of peptidyl tRNA from the chimeric ap/P into the classical A/A state upon back swivel of the 30S head [72] . Furthermore, upon translocation of peptidyl tRNA from the A to P site of the small subunit, domain IV of EF G docks into the 30S A site thus rendering the tRNA movement irreversible (Fig. 1, c and f) .

While unprecedented molecular details of the translocation mechanism have recently emerged from the structural studies and single molecule biophysical meas urements, it remains less clear how the rate of transloca tion is modulated in live cells to regulate translation elon gation. Eukaryotic translocase EF 2 was shown to be downregulated under stress conditions by phosphoryla tion [73 75] . Besides, EF 2 can be inactivated by ADP ribosylation catalyzed by diphtheria toxin [76] . A number of antibiotics hamper cell growth by hindering transloca tion in bacteria [14] . Arguably the least understood and most fascinating aspect of the translocation regulation is modulation of the translocation rate by mRNA secondary structure.

Computational analyses suggest that most, if not all, mRNAs have the propensity to form extensive intramole cular secondary structures throughout the entire sequence including the Open Reading Frame (ORF) [77] . mRNA folding results in the formation of compact structures with short end to end distances [78] . In vivo transcriptome wide RNA structure probing studies [79 84] show that mRNAs fold in live cells, at least to some degree, despite the presence of RNA helicases and other RNA binding proteins, which can disrupt RNA second ary structure. Consistent with the idea that mRNAs form extensive secondary structure in vivo, a number of struc tured mRNA elements were shown to regulate translation initiation, including bacterial riboswitches [85] , frameshift inducing hairpins and pseudoknots of eukary otic viruses [86] , Internal Ribosome Entry Sites (IRES) [87] , Iron Response Elements (IRE) in the 5′ UTR of transcripts coding for proteins involved in iron metabolism [88] , and Cap Independent Translational Enhancers (CITEs) [89] . Furthermore, protein and miRNA binding to mRNA was found to be governed by the RNA structure, which can occlude sites [90 95 ] pro viding further evidence for the importance of mRNA sec ondary structure.

Biochemical and single molecule experiments revealed that the translating ribosome is a very efficient helicase [96, 97] , which unwinds three base pairs per translocation step (Fig. 3) . The translating ribosome unfolds mRNA secondary structure because the narrow mRNA channel of the small ribosomal subunit can only BIOCHEMISTRY (Moscow) accommodate a single stranded mRNA [96, 98 101] . Consistent with the demonstrations of helicase activity of the ribosome, transcriptome wide ribosome profiling analysis demonstrates that most of the secondary struc ture elements within the coding regions of mRNAs do not influence the rate of translation elongation [102] . Most structured mRNA elements, which regulate translation, reside either in the 5′ or 3′ UTRs.

Helicase activity of the ribosome likely plays a major role in the remodeling of mRNA structure and regulating mRNA interactions with RNA binding proteins [103] . For example, mRNA translation in poly ribosomes ren ders the mRNA ORF devoid of secondary structure due to the ribosome helicase activity [103 105 ]. The pioneer round of mRNA translation by the ribosome, which dis places exon junction protein complexes (EJCs) and other proteins deposited on mRNA in the nucleus [106] , may enable mRNA folding into compact structures after ter mination of protein synthesis. Indeed, the single mole cule resolution fluorescent in situ hybridization (smFISH) and proximity ligation studies indicate lack of interactions between the distant segments of nuclear mRNAs bound with exon junction protein complexes (EJCs) [104, 107] . By contrast, the 5′ and 3′ ends of cytoplasmic mRNAs, which are not actively translated, are co localized through the formation of intramolecular secondary structure [104, 105] that tend to bring mRNA ends in close proximity [78, 108] .

Paradoxically, in spite of the ribosome helicase activ ity, certain RNA stem loop structures can induce ribo some stalling that results in accumulation of truncated polypeptides [109] and No Go mRNA decay (Fig. 3 ) [110] . Furthermore, the evolutionarily conserved mRNA stem loops and pseudoknots trigger programmed transla tion pauses [111] and stimulate -1 programmed riboso mal frameshifting (PRF), which controls expression of a number of proteins in bacteria, viruses and eukary otes [112] . In particular, -1 PRF regulates synthesis of DNA polymerase III in bacteria [113] ; HIV cytokine receptor ccr5 in higher eukaryotes [114] ; gag pol proteins in retroviruses, including Human Immunodeficiency Virus (HIV) [115] ; and C terminally extended polypro tein in coronaviruses, including SARS CoV 2, which caused the COVID 19 pandemic [116, 117] .

The mechanism of ribosome pausing induced by mRNA secondary structure is not fully understood. A number of published single molecule studies suggest that slow unwinding of a secondary structure, to which ribo some pausing is often attributed, is an unlikely explana tion of the extent of translation inhibition induced by cer tain mRNA stem loops [31, 118, 119] . Translocation through three GC base pairs is only 2 to 3 fold slower than translocation along a single stranded codon [31, 118, 119] indicating that the stability of the three base pairs adjacent to the mRNA channel has a relatively moderate effect on translocation rate. By contrast, the frameshift inducing stem loops and pseudoknots were shown to produce extended ribosome pauses [120 127 ].

It appears that rather than creating a simple road block for the ribosome, mRNA stem loops induce pro grammed ribosome pauses by making specific interac tions with the ribosome. Recent studies suggest that the frameshift inducing mRNA stem loops can perturb translation elongation by docking into the 30S A site hin dering tRNA binding [128] . Furthermore, when posi tioned at the entry of 30S mRNA channel, frameshift inducing stem loops and pseudoknots were shown to inhibit the rate of ribosomal translocation by more than one order of magnitude in a number of kinetic ensemble and single molecule experiments [120, 121, 126 129] . It has been recently demonstrated that upon encountering the mRNA secondary structure the ribosome translocates through two alternative pathways (fast and slow) [31] . Interactions of the frameshift inducing stem loops and pseudoknots with the mRNA entry channel may increase the flux through the slow pathway and thus decrease the average rate of ribosome translocation [31] .

Many of Spirin's ingenious insights into the mecha nism of ribosomal translocation were corroborated in the last few decades with advances brought by high resolu tion structures of the ribosome and single molecule bio physical experiments. Nevertheless, ribosomal transloca tion along mRNA remains one of the most fascinating steps of proteins synthesis. The complete "movie" recon structing structural rearrangements of the ribosome, EF G, and tRNA during translocation is yet to materialize. A more complete understanding of how ribosome translo cation remodels mRNA secondary structure and modu lates interactions of mRNA with many regulatory pro teins is just beginning to emerge. Mechanisms of regula tion of ribosome translocation by mRNA secondary structure await further investigation. memory of Prof. Spirin who was undergraduate research mentor to one of the authors (D. N. E.). D. N. E. is grate ful to Alexander Sergeevich Spirin for his invaluable les sons of critical thinking and research rigor. The authors thank Gregory Ballard for his comments on the manu script.

Funding. The research in the Ermolenko laboratory is supported by NIH grants R01GM099719 and R01GM132041.

Ethics declarations. The authors declare no conflicts of interest in financial or any other sphere. This article does not contain any studies involving human partici pants or animals performed by any of the authors. 

Catalysis of riboso mal translocation by sparsomycin

Properties and reg ulation of the GTPase activities of elongation factors Tu and G, and of initiation factor 2

Interaction of elongation factors EF G and EF Tu with a conserved loop in 23S RNA

The role of guanosine triphosphate in translocation reac tion catalyzed by elongation factor G

Stepwise elongation factor G promoted elongation of polypeptides on the ribosome without GTP cleavage

Elongation factor G promoted translocation and polypep tide elongation in ribosomes without GTP cleavage: use of columns with matrix bound polyuridylic acid

2011) mRNA translocation occurs during the second step of ribosomal intersubunit rotation

tRNA Translocation by the eukaryotic 80S ribosome and the impact of GTP hydrolysis

Kinetically competent intermediates in the translocation step of protein synthesis

EF G activation by phosphate analogs

EF G catalyzed translocation of anticodon stem loop analogs of transfer RNA in the ribosome

Co temporal force and fluorescence measure ments reveal a ribosomal gear shift mechanism of transla tion regulation by structured mRNAs

Translocation in protein synthesis: a hybrid structure model

Translocation makes the ribo some less compact

Structural dynamics of translating ribosomes

Structure and dynamics of a processive Brownian motor: the translating ribosome

Intermediate states in the movement of transfer RNA in the ribosome

A ratchet like inter subunit reorganization of the ribosome during transloca tion

Locking and unlock ing of ribosomal motions

Structural dynamics of the ribosome

Observation of intersubunit movement of the ribosome in solution using FRET

The antibiotic viomycin traps the ribosome in an interme diate state of translocation

Visualization of the hybrid state of tRNA binding promoted by spontaneous ratcheting of the ribosome

Structure of ratcheted ribosomes with tRNAs in hybrid states

tRNA dynamics on the ribosome during translation

Spontaneous intersubunit rotation in single ribo somes

Coupling of ribosomal L1 stalk and tRNA dynamics during translation elongation

GTP hydrolysis by IF2 guides progression of the ribosome into elongation

Domain movements of elongation factor eEF2 and the eukaryotic 80S ribosome facilitate tRNA translocation

Structures of the bacterial ribosome at 3.5 Å resolution

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

Proc. Natl. Acad. Sci. USA

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

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

Mutations in domain IV of elongation factor EF G confer 1 frameshifting

Active role of elongation factor G in maintaining the mRNA reading frame during translation

Structure of the ribosome with elon gation factor G trapped in the pretranslocation state

Following movement of domain IV of elongation factor G during ribosomal translocation

The structure of the ribosome with elongation factor G trapped in the posttranslocational state

Messenger RNA inter actions in the decoding center control the rate of transloca tion

EF G catalyzes tRNA translocation by disrupting interac tions between decoding center and codon anticodon duplex

Ensemble cryo EM uncovers inchworm like translocation of a viral IRES through the ribosome

Structures of modified eEF2 80S ribosome complexes reveal the role of GTP hydrolysis in translocation

Studies on the formation of transfer ribonucleic acid ribosome complexes. 3. The formation of peptide bonds by ribosomes in the absence of supernatant enzymes

Ribosomal protein S12 and 'non enzymatic' translocation

Interaction of SH reagents with the ribosomal 30 S subparticle and 'non enzymatic' translocation

Ribosomal proteins S12 and S13 function as control elements for translocation of the mRNA:tRNA complex

Antibiotics that bind to the A site of the large ribo somal subunit can induce mRNA translocation

Direct measurement of the mechanical work during translocation by the ribosome

Protein power strokes

Brownian ratchet mechanisms of motor proteins

Conformationally restricted elongation factor G retains GTPase activity but is inactive in translocation on the ribosome

Elongation factor G initiates translocation through a power stroke

Head swivel on the ribo some facilitates translocation by means of intra subunit tRNA hybrid sites

Phosphorylation of the elongation factor 2: the fifth Ca 2+ /calmodulin depend ent system of protein phosphorylation

Phosphorylation of elongation factor 2 by EF 2 kinase affects rate of translation

Regulation of protein synthesis at the elongation stage. New insights into the control of gene expression in eukary otes

ADP ribo sylated elongation factor 2 (ADP ribosyl EF 2) is unable to promote translocation within the ribosome

Making ends meet: new functions of mRNA secondary structure

) mRNAs and lncRNAs intrinsi cally form secondary structures with short end to end dis tances

profiling of RNA secondary structure reveals novel regula tory features

Genome wide probing of RNA structure reveals active unfolding of mRNA structures in vivo

In vivo mapping of eukaryotic RNA interactomes reveals principles of higher order organization and regula tion

RNA Duplex map in living cells reveals high er order transcriptome structure

Global mapping of human RNA-RNA interactions

COMRADES deter mines in vivo RNA structures and interactions

The structural and functional diversity of metabolite binding riboswitches

Frameshifting RNA pseudoknots: structure and mechanism

The genetic code as expressed through relationships between mRNA structure and protein function

The family of iron responsive RNA structures regulated by changes in cellular iron and oxygen

3′ cap independent translation enhancers of plant viruses

Effect of target sec ondary structure on RNAi efficiency

Efficient siRNA selection using hybridization thermodynamics

Fundamental differ ences in the equilibrium considerations for siRNA and antisense oligodeoxynucleotide design

The impact of target site accessibility on the design of effective siRNAs

Predicting in vivo binding sites of RNA binding proteins using mRNA secondary structure

Finding the target sites of RNA binding proteins

mRNA helicase activity of the ribosome

Following translation by single ribosomes one codon at a time

The path of messenger RNA through the ribo some

Transition of the mRNA sequence downstream from the initiation codon into a single stranded conforma tion is strongly promoted by binding of the initiator tRNA

Secondary struc ture of the ribosome binding site determines translational efficiency: a quantitative analysis

RNA folding kinetics regulates translation of phage MS2 maturation gene

Secondary Structure across the bacte rial transcriptome reveals versatile roles in mRNA regula tion and function

The landscape of eukaryotic mRNPs

Spatial organization of single mRNPs at different stages of the gene expression pathway

mRNP architecture in translating and stress conditions reveals an ordered path way of mRNP compaction

The pio neer round of translation: features and functions

Higher order organization prin ciples of pre translational mRNPs

Heterogeneous dynamics of protein-RNA interactions across transcriptome derived messenger RNA popula tions

Ribosome excursions during mRNA translocation mediate broad branching of frameshift pathways

Endonucleolytic cleav age of eukaryotic mRNAs with stalls in translation elonga tion

The signal recog nition particle receptor alpha subunit assembles co trans lationally on the endoplasmic reticulum membrane during an mRNA encoded translation pause in vitro

Changed in translation: mRNA recoding by 1 pro grammed ribosomal frameshifting

Translational frameshifting generates the gamma subunit of DNA poly merase III holoenzyme

Ribosomal frameshifting in the CCR5 mRNA is regulated by miRNAs and the NMD pathway

Characterization of riboso mal frameshifting in HIV 1 gag pol expression

Structural and functional con servation of the programmed 1 ribosomal frameshift sig nal of SARS coronavirus 2 (SARS CoV 2)

Programmed 1 ribosomal frameshifting in coronaviruses: a therapeutic target

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

Dynamics of translation by single ribosomes through mRNA secondary structures

Dynamic pathways of 1 translational frameshifting

A frameshifting stimu latory stem loop destabilizes the hybrid state and impedes ribosomal translocation

Ribosomal movement impeded at a pseudoknot required for frameshifting

Ribosomal pausing during translation of an RNA pseudoknot

Kinetics of ribosomal pausing during programmed 1 translational frameshifting

Ribosomal pausing at a frameshifter RNA pseudoknot is sensitive to reading phase but shows little correlation with frameshift efficiency

Programmed 1 frameshifting by kinetic partitioning during impeded translocation

Conditional switch between frameshifting regimes upon translation of dnaX mRNA

2020) mRNA stem loops can pause the ribosome by hindering A site tRNA binding

The energy landscape of 1 ribosomal frameshift ing