Brightness-gated two-color coincidence detection unravels two distinct mechanisms in bacterial protein translation initiation.
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ABSTRACT:
SUBMITTER: Hofig H
PROVIDER: S-EPMC6897966 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:BackgroundThe selection and regulation of individual mRNAs for translation initiation from a competing pool of mRNA are poorly understood processes. The closed loop complex, comprising eIF4E, eIF4G and PABP, and its regulation by 4E-BPs are perceived to be key players. Using RIP-seq, we aimed to evaluate the role in gene regulation of the closed loop complex and 4E-BP regulation across the entire yeast transcriptome.ResultsWe find that there are distinct populations of mRNAs with coherent properties: one mRNA pool contains many ribosomal protein mRNAs and is enriched specifically with all of the closed loop translation initiation components. This class likely represents mRNAs that rely heavily on the closed loop complex for protein synthesis. Other heavily translated mRNAs are apparently under-represented with most closed loop components except Pab1p. Combined with data showing a close correlation between Pab1p interaction and levels of translation, these data suggest that Pab1p is important for the translation of these mRNAs in a closed loop independent manner. We also identify a translational regulatory mechanism for the 4E-BPs; these appear to self-regulate by inhibiting translation initiation of their own mRNAs.ConclusionsOverall, we show that mRNA selection for translation initiation is not as uniformly regimented as previously anticipated. Components of the closed loop complex are highly relevant for many mRNAs, but some heavily translated mRNAs interact poorly with this machinery. Therefore, alternative, possibly Pab1p-dependent mechanisms likely exist to load ribosomes effectively onto mRNAs. Finally, these studies identify and characterize a complex self-regulatory circuit for the yeast 4E-BPs.
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Project description:Store-operated calcium entry is an almost ubiquitous signaling pathway in eukaryotic cells. The plasma membrane store-operated channels are comprised of subunits of the recently discovered Orai proteins, the major one being Orai1.We have discovered that native Orai1, as well as expressed Orai1, exists in two forms in similar quantities: a longer form (Orai1?) of approximately 33 kDa, and a shorter form (Orai1?) of approximately 23 kDa. The Orai1? form arises from alternative translation initiation from a methionine at position 64, and possibly also 71, in the longer Orai1? form. In the sequence upstream of the initiation site of Orai1?, there is a poly-arginine sequence previously suggested to be involved in interaction of Orai1 with plasma membrane phosphatidylinositol-4,5-bisphosphate. The loss of this phospholipid binding domain would be expected to influence the mobility of Orai1 protein in the plasma membrane. Indeed, experiments utilizing fluorescence recovery after photobleaching (FRAP) revealed that the recovery half-time for Orai1? was significantly faster than for Orai1?. Since Orai1 must diffuse to sites of interaction with the Ca(2+) sensor, STIM1, these two mobilities might provide for efficient recruitment of Orai1 subunits to sites of store-operated Ca(2+) entry during agonist-induced Ca(2+) signaling.
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Project description:In higher eukaryotes, eIF4A, eIF4E and eIF4G homologues interact to enable mRNA recruitment to the ribosome. eIF4G acts as a scaffold for these interactions and also interacts with other proteins of the translational machinery. Trypanosomatid protozoa have multiple homologues of eIF4E and eIF4G and the precise function of each remains unclear. Here, 2 previously described eIF4G homologues, EIF4G3 and EIF4G4, were further investigated. In vitro, both homologues bound EIF4AI, but with different interaction properties. Binding to distinct eIF4Es was also confirmed; EIF4G3 bound EIF4E4 while EIF4G4 bound EIF4E3, both these interactions required similar binding motifs. EIF4G3, but not EIF4G4, interacted with PABP1, a poly-A binding protein homolog. Work in vivo with Trypanosoma brucei showed that both EIF4G3 and EIF4G4 are cytoplasmic and essential for viability. Depletion of EIF4G3 caused a rapid reduction in total translation while EIF4G4 depletion led to changes in morphology but no substantial inhibition of translation. Site-directed mutagenesis was used to disrupt interactions of the eIF4Gs with either eIF4E or eIF4A, causing different levels of growth inhibition. Overall the results show that only EIF4G3, with its cap binding partner EIF4E4, plays a major role in translational initiation.
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Project description:Translation initiation in human mitochondria relies upon specialized mitoribosomes and initiation factors, mtIF2 and mtIF3, which have diverged from their bacterial counterparts. Here we report two distinct mitochondrial pre-initiation assembly steps involving those factors. Single-particle cryo-EM revealed that in the first step, interactions between mitochondria-specific protein mS37 and mtIF3 keep the small mitoribosomal subunit in a conformation favorable for a subsequent accommodation of mtIF2 in the second step. Combination with fluorescence cross-correlation spectroscopy analyses suggests that mtIF3 promotes complex assembly without mRNA or initiator tRNA binding, where exclusion is achieved by the N-terminal and C-terminal domains of mtIF3. Finally, the association of large mitoribosomal subunit is required for initiator tRNA and leaderless mRNA recruitment to form a stable initiation complex. These data reveal fundamental aspects of mammalian protein synthesis that are specific to mitochondria.
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Project description:It is generally believed that prokaryotic translation is initiated by the interaction between the Shine-Dalgarno (SD) sequence in the 5' UTR of an mRNA and the anti-SD sequence in the 3' end of a 16S ribosomal RNA. However, there are two exceptional mechanisms, which do not require the SD sequence for translation initiation: one is mediated by a ribosomal protein S1 (RPS1) and the other used leaderless mRNA that lacks its 5' UTR. To understand the evolutionary changes of the mechanisms of translation initiation, we examined how universal the SD sequence is as an effective initiator for translation among prokaryotes. We identified the SD sequence from 277 species (249 eubacteria and 28 archaebacteria). We also devised an SD index that is a proportion of SD-containing genes in which the differences of GC contents are taken into account. We found that the SD indices varied among prokaryotic species, but were similar within each phylum. Although the anti-SD sequence is conserved among species, loss of the SD sequence seems to have occurred multiple times, independently, in different phyla. For those phyla, RPS1-mediated or leaderless mRNA-used mechanisms of translation initiation are considered to be working to a greater extent. Moreover, we also found that some species, such as Cyanobacteria, may acquire new mechanisms of translation initiation. Our findings indicate that, although translation initiation is indispensable for all protein-coding genes in the genome of every species, its mechanisms have dynamically changed during evolution.
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Project description:Eukaryotic mRNAs often recruit ribosomal subunits some distance upstream of the initiation codon; however, the mechanisms by which they reach the initiation codon remain to be fully elucidated. Although scanning is a widely accepted model, evidence for alternative mechanisms has accumulated. We previously suggested that this process may involve tethering of ribosomal complexes to the mRNA, in which the intervening mRNA is bypassed, or clustering, in which the initiation codon is reached by dynamic binding and release of ribosomal subunits at internal sites. The present studies tested the feasibility of these ideas by using model mRNAs and revealed that translation efficiency varied with the distance between the site of ribosomal recruitment and the initiation codon. The present studies also showed that translation could initiate efficiently at AUG codons located upstream of an internal site. These observations are consistent with ribosomal tethering at the cap structure and clustering at internal sites.
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Project description:Eubacterial translation initiation involves assembly of tRNAfMet, mRNA, initiation factors (IFs) and 30S ribosome in a 30S pre-initiation complex (30S pre-IC), which rearranges and joins 50S ribosome to form 70S IC. Upon releasing IFs, 70S IC becomes elongation-competent 70S. The direct recruitment of initiator tRNA (tRNAfMet) into the ribosomal P-site, crucial in accurate initiation of translation, is attributed to two conserved features of tRNAfMet: (i) formylation of amino acid attached to it and, (ii) the presence of three consecutive G-C base pairs (3GC base pairs) in the anticodon stem. However, the precise roles of these two conserved features of tRNAfMet during the various steps of initiation remain unclear. Using natural and engineered tRNAs, we show that the 3GC pairs license tRNAfMet transitions from 30S to 70S IC and then to elongation-competent 70S by release of IF3. Of the 3GC pairs, the middle GC pair (G30-C40), or merely G30 (in a specific context) suffices in this role and is essential for the sustenance of Escherichia coli. Furthermore, rescue of formylase deficient E. coli by overproduced tRNAfMet reveals that the feature of formylation licenses initial targeting of tRNAfMet to 30S ribosome
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Project description:MHC class I molecules present a comprehensive mixture of peptides on the cell surface for immune surveillance. The peptides represent the intracellular protein milieu produced by translation of endogenous mRNAs. Unexpectedly, the peptides are encoded not only in conventional AUG initiated translational reading frames but also in alternative cryptic reading frames. Here, we analyzed how ribosomes recognize and use cryptic initiation codons in the mRNA. We find that translation initiation complexes assemble at non-AUG codons but differ from canonical AUG initiation in response to specific inhibitors acting within the peptidyl transferase and decoding centers of the ribosome. Thus, cryptic translation at non-AUG start codons can utilize a distinct initiation mechanism which could be differentially regulated to provide peptides for immune surveillance.
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Project description:In prokaryotic systems, the initiation phase of protein synthesis is governed by the presence of initiation factors that guide the transition of the small ribosomal subunit (30S) from an unlocked preinitiation complex (30S preIC) to a locked initiation complex (30SIC) upon the formation of a correct codon-anticodon interaction in the peptidyl (P) site. Biochemical and structural characterization of GE81112, a translational inhibitor specific for the initiation phase, indicates that the main mechanism of action of this antibiotic is to prevent P-site decoding by stabilizing the anticodon stem loop of the initiator tRNA in a distorted conformation. This distortion stalls initiation in the unlocked 30S preIC state characterized by tighter IF3 binding and a reduced association rate for the 50S subunit. At the structural level we observe that in the presence of GE81112 the h44/h45/h24a interface, which is part of the IF3 binding site and forms ribosomal intersubunit bridges, preferentially adopts a disengaged conformation. Accordingly, the findings reveal that the dynamic equilibrium between the disengaged and engaged conformations of the h44/h45/h24a interface regulates the progression of protein synthesis, acting as a molecular switch that senses and couples the 30S P-site decoding step of translation initiation to the transition from an unlocked preIC to a locked 30SIC state.
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