Ribosome rearrangements at the onset of translational bypassing.

Bypassing is a recoding event that leads to the translation of two distal open reading frames into a single polypeptide chain. We present the structure of a translating ribosome stalled at the bypassing take-off site of gene 60 of bacteriophage T4. The nascent peptide in the exit tunnel anchors the P-site peptidyl-tRNAGly to the ribosome and locks an inactive conformation of the peptidyl transferase center (PTC). The mRNA forms a short dynamic hairpin in the decoding site. The ribosomal subunits adopt a rolling conformation in which the rotation of the small subunit around its long axis causes the opening of the A-site region. Together, PTC conformation and mRNA structure safeguard against premature termination and read-through of the stop codon and reconfigure the ribosome to a state poised for take-off and sliding along the noncoding mRNA gap

A switch from α‐helical to β‐strand conformation during co‐translational protein folding

Cellular proteins begin to fold as they emerge from the ribosome.The folding landscape of nascent chains is not only shaped by theiramino acid sequence but also by the interactions with the ribo-some. Here, we combine biophysical methods with cryo-EM struc-ture determination to show that folding of aβ-barrel proteinbegins with formation of a dynamicα-helix inside the ribosome. Asthe growing peptide reaches the end of the tunnel, the N-terminalpart of the nascent chain refolds to aβ-hairpin structure thatremains dynamic until its release from the ribosome. Contactswith the ribosome and structure of the peptidyl transferase centerdepend on nascent chain conformation. These results indicate thatproteins may start out asα-helices inside the tunnel and switchinto their native folds only as they emerge from the ribosome.Moreover, the correlation of nascent chain conformations withreorientation of key residues of the ribosomal peptidyl-transferasecenter suggest that protein folding could modulate ribosome activity

Recognition of aminoacyl-tRNA: a common molecular mechanism revealed by cryo-EM

The accuracy of ribosomal translation is achieved by an initial selection and a proofreading step, mediated by EF-Tu, which forms a ternary complex with aminoacyl(aa)-tRNA. To study the binding modes of different aa-tRNAs, we compared cryo-EM maps of the kirromycin-stalled ribosome bound with ternary complexes containing Phe-tRNAPhe, Trp-tRNATrp, or Leu-tRNALeuI. The three maps suggest a common binding manner of cognate aa-tRNAs in their specific binding with both the ribosome and EF-Tu. All three aa-tRNAs have the same ‘loaded spring' conformation with a kink and twist between the D-stem and anticodon stem. The three complexes are similarly integrated in an interaction network, extending from the anticodon loop through h44 and protein S12 to the EF-Tu-binding CCA end of aa-tRNA, proposed to signal cognate codon–anticodon interaction to the GTPase centre and tune the accuracy of aa-tRNA selection

The Cryo-EM Structure of a Complete 30S Translation Initiation Complex from Escherichia coli

Formation of the 30S initiation complex (30S IC) is an important checkpoint in regulation of gene expression. The selection of mRNA, correct start codon, and the initiator fMet-tRNAfMet requires the presence of three initiation factors (IF1, IF2, IF3) of which IF3 and IF1 control the fidelity of the process, while IF2 recruits fMet-tRNAfMet. Here we present a cryo-EM reconstruction of the complete 30S IC, containing mRNA, fMet-tRNAfMet, IF1, IF2, and IF3. In the 30S IC, IF2 contacts IF1, the 30S subunit shoulder, and the CCA end of fMet-tRNAfMet, which occupies a novel P/I position (P/I1). The N-terminal domain of IF3 contacts the tRNA, whereas the C-terminal domain is bound to the platform of the 30S subunit. Binding of initiation factors and fMet-tRNAfMet induces a rotation of the head relative to the body of the 30S subunit, which is likely to prevail through 50S subunit joining until GTP hydrolysis and dissociation of IF2 take place. The structure provides insights into the mechanism of mRNA selection during translation initiation

Molecular dynamics of ribosomal elongation factors G and Tu

Translation on the ribosome is controlled by external factors. During polypeptide lengthening, elongation factors EF-Tu and EF-G consecutively interact with the bacterial ribosome. EF-Tu binds and delivers an aminoacyl-tRNA to the ribosomal A site and EF-G helps translocate the tRNAs between their binding sites after the peptide bond is formed. These processes occur at the expense of GTP. EF-Tu:tRNA and EF-G are of similar shape, share a common binding site, and undergo large conformational changes on interaction with the ribosome. To characterize the internal motion of these two elongation factors, we used 25 ns long all-atom molecular dynamics simulations. We observed enhanced mobility of EF-G domains III, IV, and V and of tRNA in the EF-Tu:tRNA complex. EF-Tu:GDP complex acquired a configuration different from that found in the crystal structure of EF-Tu with a GTP analogue, showing conformational changes in the switch I and II regions. The calculated electrostatic properties of elongation factors showed no global similarity even though matching electrostatic surface patches were found around the domain I that contacts the ribosome, and in the GDP/GTP binding region

SecM-Stalled Ribosomes Adopt an Altered Geometry at the Peptidyl Transferase Center

A structure of a ribosome stalled during translation of the SecM peptide provides insight into the mechanism by which the large subunit active site is inactivated

Cotranslational protein assembly imposes evolutionary constraints on homomeric proteins

Cotranslational protein folding can facilitate rapid formation of functional structures. However, it might also cause premature assembly of protein complexes, if two interacting nascent chains are in close proximity. By analyzing known protein structures, we show that homomeric protein contacts are enriched towards the C-termini of polypeptide chains across diverse proteomes. We hypothesize that this is the result of evolutionary constraints for folding to occur prior to assembly. Using high-throughput imaging of protein homomers in vivo in E. coli and engineered protein constructs with N- and C-terminal oligomerization domains, we show that, indeed, proteins with C-terminal homomeric interface residues consistently assemble more efficiently than those with N-terminal interface residues. Using in vivo, in vitro and in silico experiments, we identify features that govern successful assembly of homomers, which have implications for protein design and expression optimization

Running title: Functional characterization of putative CTDK-1 proteins in Aspergillus.

58 p.-8 fig.The genus Aspergillus includes industrially, medically and agriculturally important species. All of them, as do fungi in general, disperse to new niches principally by means of asexual spores. Regarding the genetic/molecular control of asexual development, Aspergillus nidulans is the main reference. In this species, two pathways control the production of conidiophores, the structures bearing asexual spores (conidia). The Upstream Developmental Activation (UDA) pathway transduces environmental signals, determining whether the Central Developmental Pathway (CDP) and the required morphological changes are induced. The transcriptional regulator BrlA links both pathways as loss-of-function mutations in flb (UDA) genes block brlA transcription and, consequently, conidiation. However, the aconidial phenotype of specific flb mutants is reverted under salt-stress conditions. Previously, we generated a collection of ΔflbB mutants unable to conidiate on culture medium supplemented with NaH2PO4 (0.65M). Here, we identified a Gly347Stop mutation within flpA as responsible for the FLIP57 phenotype. The putative cyclin FlpA and the remaining putative components of the C-terminal domain kinase-1 (CTDK-1) complex are necessary for proper germination, growth and developmental patterns in both A. nidulans and A. fumigatus. Cellular localization and functional interdependencies of the three proteins are also analyzed. Overall, this work links the putative CTDK-1 complex of aspergilli with growth and developmental control.Work at O.E. s lab has been supported by UPV/EHU (GIU19/014 to O.E.) and the Basque Government (PIBA-PUE PIBA_2020_1_0032; Elkartek KK-2021/43 and KK 2022/00107; and GIC IT1662-22, to O.E.). Work at CIB Margarita Salas-CSIC has been supported by MICIU/AEI (RTI2018-094263-B-100, to E.A.E.). A.O. hold a Margarita Salas grant (MARSA21/69), funded by Next-Generation EU, at the UPV/EHU. A.F was a degree student with a collaboration grant by the Spanish Ministry of Education (21/19070). Z.A was a Master Thesis student at O.E.´s lab, held an Ikertalent Fellow funded by the Basque Government (PIF21/003) at the Basque Culinary Center, and is now a PhD student at O.E.´s lab with funds of the KK 2022/00107 project. Work at J.R.F.´s lab has been supported by NIH grants R01-AI158442 and R01-AI143197.Peer reviewe