Late steps in bacterial translation initiation visualized using time-resolved cryo-EM.

Genetic studies in yeast have shown that the translation initiation factor eIF5 plays an important role in the selection of the AUG start codon. In order to ensure translation fidelity, the hydrolysis of GTP bound to the 40S preinitiation complex (40S.Met-tRNA(i).eIF2.GTP), promoted by eIF5, must occur only when the complex has selected the AUG start codon. However, the mechanism that prevents the eIF5-promoted GTP hydrolysis, prior to AUG selection by the ribosomal machinery, is not known. In this work, we show that the presence of initiation factors eIF1, eIF1A and eIF3 in the 40S preinitiation complex (40S.eIF1.eIF1A.eIF3.Met-tRNA(i).eIF2.GTP) and the subsequent binding of the preinitiation complex to eIF4F bound at the 5'-cap structure of mRNA are necessary for preventing eIF5-promoted hydrolysis of GTP in the 40S preinitiation complex. This block in GTP hydrolysis is released upon AUG selection by the 40S preinitiation complex. These results, taken together, demonstrate the biochemical requirements for regulation of GTP hydrolysis and its coupling to the AUG selection process during translation initiation.

Bacterial translation initiation factor IF2 promotes ribosomal subunit association, recruitment, and binding of fMet-tRNA to the ribosomal P-site and initiation dipeptide formation. Here, we present the solution structures of GDP-bound and apo-IF2-G2 of Bacillus stearothermophilus and provide evidence that this isolated domain binds the 50 S ribosomal subunit and hydrolyzes GTP. Differences between the free and GDP-bound structures of IF2-G2 suggest that domain reorganization within the G2-G3-C1 regions underlies the different structural requirements of IF2 during the initiation process. However, these structural signals are unlikely forwarded from IF2-G2 to the C-terminal fMet-tRNA binding domain (IF2-C2) because the connected IF2-C1 and IF2-C2 modules show completely independent mobility, indicating that the bacterial interdomain connector lacks the rigidity that was found in the archaeal IF2 homolog aIF5B.

Kinetic Checkpoint at a Late Step in Translation Initiation

The influence of the rare initiation triplet AUU on mRNA translation was investigated by comparing the activity of two pairs of model mRNAs that differ in the length of Shine-Dalgarno and spacer sequences. Irrespective of the initiation triplet (AUG or AUU), all mRNAs had similar template activity in vitro, but translation of AUU mRNAs depended more on initiation factor (IF) 2 and less on IF3 than that of AUG mRNAs. Increasing the IF3/ribosome ratio from 2 to 10 progressively inhibited the AUU mRNAs and abolished their capacity to compete for translating ribosomes with other mRNAs but did not affect activity of the AUG mRNAs. The effects induced by IF3 are from its different influence on on- and off-rates of the transition 30S preinitiation complex<==>30S initiation complex; depending on the nature of the initiation triplet (AUG or AUU) of the mRNA, IF3 shifts the position of equilibrium toward binding or dissociation of fMet-tRNA, respectively. Stimulation of fMet-tRNA binding and dissociation yields superimposable IF3 titration curves that saturate at an IF3/30S ratio of approximately 1, indicating that the data are from the interaction of one molecule of IF3 with the same 30S binding site. Both effects are either lost or strongly reduced with 30S mutants defective in IF3 binding. Translational repression of AUU mRNAs by IF3 is from the factor-dependent dissociation of fMet-tRNA from 30S subunits, which becomes relevant when excess IF3 interferes with the formation of 70S initiation complex, presumably by interacting with 50S subunit.

During formation of the transcription-competent open complex (RPo) by bacterial RNA polymerases (RNAPs), transient intermediates pile up before overcoming a rate-limiting step. Structural descriptions of these interconversions in real time are unavailable. To address this gap, here we use time-resolved cryogenic electron microscopy (cryo-EM) to capture four intermediates populated 120 ms or 500 ms after mixing Escherichia coli σ70-RNAP and the λPR promoter. Cryo-EM snapshots revealed that the upstream edge of the transcription bubble unpairs rapidly, followed by stepwise insertion of two conserved nontemplate strand (nt-strand) bases into RNAP pockets. As the nt-strand 'read-out' extends, the RNAP clamp closes, expelling an inhibitory σ70 domain from the active-site cleft. The template strand is fully unpaired by 120 ms but remains dynamic, indicating that yet unknown conformational changes complete RPo formation in subsequent steps. Given that these events likely describe DNA opening at many bacterial promoters, this study provides insights into how DNA sequence regulates steps of RPo formation.

Two genes specifying model mRNAs of minimal size and coding capacity, with or without the Shine-Dalgarno (SD) sequence, were assembled, cloned, and transcribed in high yields. These mRNAs, as well as synthetic polynucleotides, phage MS2 RNA, and a deoxyoctanucleotide complementary to the 3' end of 16S rRNA were used to study the mechanism of translation initiation in vitro. Escherichia coli 30S ribosomal subunits interact with all these nucleic acids, albeit with different affinities; the affinity for the mRNA with the SD sequence (Ka approximately 2 x 10(7) M-1) is more than an order of magnitude higher than that for the mRNA lacking this sequence. The initiation factors are equally required, regardless of the presence of the SD sequence, for 30S and 70S initiation complex formation and for mRNA translation, but the initiation factors do not affect the SD interaction or the binding of the mRNAs to the ribosomes. The SD interaction is also mechanistically irrelevant for 30S initiation complex formation and is not essential for translation in vitro or for the selection of the mRNA reading frame. It is suggested that the function of the SD interaction is to ensure a high concentration of the initiation triplet near the ribosomal peptidyl-tRNA binding site, whereas the selection of the translational start is achieved kinetically, under the influence of the initiation factors, during decoding of the initiator tRNA.

When the ribosome encounters a stop codon, it recruits a release factor (RF) to hydrolyze the ester bond between the peptide chain and tRNA. This chapter discusses the role of the compact structures of RF1 and RF2 for fast and accurate stop-codon recognition in translation termination. It utilizes time-resolved cryogenic electron microscopy (cryo-EM) to visualize transient compact forms of RF1 and RF2 at 3.5 and 4 Å resolution, respectively, in the codon-recognizing ribosome complex on the native pathway. Cryo-EM showed that ribosome-bound RFs have extended structures, suggesting that RFs are compact when entering the ribosome and then extend their structures upon stop codon recognition. Most intracellular functions are carried out by proteins, assembled as chains of peptide-bond linked amino acid residues on large ribonucleoprotein particles called ribosomes. The chapter suggest that the selective advantage of the compact RF-form is that it allows for rapid factor binding into and dissociation from an accuracy maximizing pre-accommodation state.

Most eukaryotic initiation factors (eIFs) are required for internal translation initiation at the internal ribosome entry site (IRES) of picornaviruses. eIF4B is incorporated into ribosomal 48S initiation complexes with the IRES RNA of foot-and-mouth disease virus (FMDV). In contrast to the weak interaction of eIF4B with capped cellular mRNAs and its release upon entry of the ribosomal 60S subunit, eIF4B remains tightly associated with the FMDV IRES during formation of complete 80S ribosomes. Binding of eIF4B to the IRES is energy dependent, and binding of the small ribosomal subunit to the IRES requires the previous energy-dependent association of initiation factors with the IRES. The interaction of eIF4B with the IRES in 48S and 80S complexes is independent of the location of the initiator AUG and thus independent of the mechanism by which the small ribosomal subunit is placed at the actual start codon, either by direct internal ribosomal entry or by scanning. eIF4B does not greatly rearrange its binding to the IRES upon entry of the ribosomal subunits, and the interaction of eIF4B with the IRES is independent of the polypyrimidine tract-binding protein, which enhances FMDV translation.

The rate of protein synthesis in skeletal muscle is greatly decreased in response to diabetes and starvation. Analysis of polyribosome profiles indicates that polypeptide-chain initiation is impaired under these conditions. To identify the step in initiation that is affected, we assayed the incorporation of [35S]methionyl-tRNAfMet into [35S]methionyl-tRNAfMet . 40S-ribosomal-subunit initiation complexes in cell-free extracts based on postmitochondrial supernatants prepared from gastrocnemius muscle. Extracts from either starved or diabetic rats were 30-40% less active in forming these complexes compared with those derived from fed or insulin-maintained controls respectively. This change could be reversed by treatment of either starved or diabetic rats with insulin in vivo 30 min before death. Formation of 40S initiation complexes by extracts from either fed or starved rats could be stimulated by the addition of exogenous purified initiation factor eIF-2, but extracts from starved or diabetic rats were more sensitive than controls to stimulation by low concentrations of the factor. These results provide evidence for the acute regulation by insulin of protein synthesis in skeletal muscle at the level of polypeptide-chain initiation, and suggest that in this tissue, as in certain other eukaryotic systems, control of initiation appears to be mediated by changes in the activity of initiation factor eIF-2.

Ribosomal stalling induces the ribosome-associated quality control (RQC) pathway targeting aberrant polypeptides. RQC is initiated by K63-polyubiquitination of ribosomal protein uS10 located at the mRNA entrance of stalled ribosomes by the E3 ubiquitin ligase ZNF598 (Hel2 in yeast). Ubiquitinated ribosomes are dissociated by the ASC-1 complex (ASCC) (RQC-Trigger (RQT) complex in yeast). A cryo-EM structure of the ribosome-bound RQT complex suggested the dissociation mechanism, in which the RNA helicase Slh1 subunit of RQT (ASCC3 in mammals) applies a pulling force on the mRNA, inducing destabilizing conformational changes in the 40S subunit, whereas the collided ribosome acts as a wedge, promoting subunit dissociation. Here, using an in vitro reconstitution approach, we found that ribosomal collision is not a strict prerequisite for ribosomal ubiquitination by ZNF598 or for ASCC-mediated ribosome release. Following ubiquitination by ZNF598, ASCC efficiently dissociated all polysomal ribosomes in a stalled queue, monosomes assembled in RRL, in vitro reconstituted 80S elongation complexes in pre- and post-translocated states, and 48S initiation complexes, as long as such complexes contained ≥ 30–35 3′-terminal mRNA nt. downstream from the P site and sufficiently long ubiquitin chains. Dissociation of polysomes and monosomes both involved ribosomal splitting, enabling Listerin-mediated ubiquitination of 60S-associated nascent chains.

Inhibitory codon pairs and poly(A) tracts within the translated mRNA cause ribosome stalling and reduce protein output. The molecular mechanisms that drive these stalling events, however, are still unknown. Here, we use a combination of invitro biochemistry, ribosome profiling, and cryo-EM to define molecular mechanisms that lead to these ribosome stalls. First, we use an invitro reconstituted yeast translation system to demonstrate that inhibitory codon pairs slow elongation rates which are partially rescued by increased tRNA concentration or by an artificial tRNA not dependent on wobble base-pairing. Ribosome profiling data extend these observations by revealing that paused ribosomes with empty A sites are enriched on these sequences. Cryo-EM structures of stalled ribosomes provide a structural explanation for the observed effects by showing decoding-incompatible conformations of mRNA in the A sites of all studied stall- and collision-inducing sequences. Interestingly, in the case of poly(A) tracts, the inhibitory conformation of the mRNA in the A site involves a nucleotide stacking array. Together, these data demonstrate a novel mRNA-induced mechanisms of translational stalling in eukaryotic ribosomes.

Chromosomal replication initiation requires the regulated formation of dynamic higher order complexes. Escherichia coli ATP-DnaA forms a specific multimer on oriC, resulting in DNA unwinding and DnaB helicase loading. DiaA, a DnaA-binding protein, directly stimulates the formation of ATP-DnaA multimers on oriC and ensures timely replication initiation. In this study, DnaA Phe-46 was identified as the crucial DiaA-binding site required for DiaA-stimulated ATP-DnaA assembly on oriC. Moreover, we show that DiaA stimulation requires only a subgroup of DnaA molecules binding to oriC, that DnaA Phe-46 is also important in the loading of DnaB helicase onto the oriC-DnaA complexes, and that this process also requires only a subgroup of DnaA molecules. Despite the use of only a DnaA subgroup, DiaA inhibited DnaB loading on oriC-DnaA complexes, suggesting that DiaA and DnaB bind to a common DnaA subgroup. A cellular factor can relieve the DiaA inhibition, allowing DnaB loading. Consistently, DnaA F46A caused retarded initiations in vivo in a DiaA-independent manner. It is therefore likely that DiaA dynamics are crucial in the regulated sequential progress of DnaA assembly and DnaB loading. We accordingly propose a model for dynamic structural changes of initial oriC complexes loading DiaA or DnaB helicase.

The 30S initiation complex, formed with the 30S ribosomal subunit, mRNA, and fMet-tRNA, has been shown by kinetic analysis with limiting concentrations of Escherichia coli ribosomes to be an obligatory intermediate in the formation of the 70S initiation complex. The formation of the 70S initiation complex began with an induction period and was proportional to the concentration of the 30S complex, which rapidly rose to a peak. The entire time course of the sequential pseudo-first-order, second-order reaction was reproduced accurately by the overall rate expression, in which we used rate constants that were determined by carrying out 30S and 70S complex formation separately. By using limiting concentrations of mRNA, we showed that phage MS2 RNA contained no specific signal that enhanced its rate of 30S complex formation with E. coli ribosomes and initiation factors; the pseudo-first-order rate constants obtained with poly(A3C9G1U1), poly(C15G1U4), and poly(G1U3) were 12-45 times higher than that with MS2 RNA. The observation that the rate constants for binding of fMet-tRNA and AcPhe-tRNA with a given synthetic RNA were comparable indicated that the initiator codon is recognized only indirectly through the initiator tRNA.

Furvina®, also denominated G1 (MW 297), is a synthetic nitrovinylfuran [2-bromo-5-(2-bromo-2-nitrovinyl)-furan] antibiotic with a broad antimicrobial spectrum. An ointment (Dermofural®) containing G1 as the only active principle is currently marketed in Cuba and successfully used to treat dermatological infections. Here we describe the molecular target and mechanism of action of G1 in bacteria and demonstrate that in vivo G1 preferentially inhibits protein synthesis over RNA, DNA and cell wall synthesis. Furthermore, we demonstrate that G1 targets the small ribosomal subunit, binds at or near the P-decoding site and inhibits its function interfering with the ribosomal binding of fMet-tRNA during 30S initiation complex (IC) formation ultimately inhibiting translation. Notably, this G1 inhibition displays a bias for the nature (purine vs. pyrimidine) of the 3′-base of the codon, occurring efficiently only when the mRNA directing 30S IC formation and translation contains the canonical AUG initiation triplet or the rarely found AUA triplet, but hardly occurs when the mRNA start codon is either one of the non-canonical triplets AUU or AUC. This codon discrimination by G1 is reminiscent, though of opposite type of that displayed by IF3 in its fidelity function, and remarkably does not occur in the absence of this factor.

Non-canonical Binding Site for Bacterial Initiation Factor 3 on the Large Ribosomal Subunit