During the last few decades, the ribosome has been regarded primarily as a major cell player
devoted to the catalysis of protein biosynthesis during translation [1-5]. It is therefore not
surprising that several processes related to translation exploit the ribosome as a central hub. For
instance, it is well-known that many events related to translational regulation are mediated by
interactions between the ribosome and initiation, elongation or termination factors [6-9]. In
addition, the ribosome is involved in mRNA-code recognition and proofreading [10-12] as well
as in the control of translation rates via interactions with mRNA codons bearing high- and lowfrequency
[13-15] and associated with variable tRNA abundance within the translation machinery
[16-18]. Interestingly, the ribosome also assists de novo protein structure formation by minimizing
cotranslational aggregation, thus increasing the yield of native-protein production [19,20]. The
latter event, however, has not been shown to require -- or even involve -- direct interactions between
the ribosome and the nascent protein chain. A notable exception is that of nascent chains bearing
either N-terminal signal sequences or translational-arrest tags. These proteins are known to establish
short- or long-term contacts with various regions of the ribosome during translation [21-25]. In
summary, until recently very little knowledge has been available about direct contacts between the
ribosome and nascent polypeptides and proteins that do not carry signal or arrest sequences. Studies
based on fluorescence depolarization in the frequency domain [26] and NMR spectroscopy [27-30]
provided interesting data that are consistent with, but do not unequivocally establish, the presence
of these interactions.
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ROSE: a deep learning based framework for predicting ribosome stalling
We present a deep learning based framework, called ROSE, to accurately predict ribosome stalling events in translation elongation from coding sequences based on high-throughput ribosome profiling data. Our validation results demonstrate the superior performance of ROSE over conventional prediction models. ROSE provides an effective index to estimate the likelihood of translational pausing at codon resolution and understand diverse putative regulatory factors of ribosome stalling. Also, the ribosome stalling landscape computed by ROSE can recover the functional interplay between ribosome stalling and cotranslational events in protein biogenesis, including protein targeting by the signal recognition particle (SRP) and protein secondary structure formation.
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- NSF-PAR ID:
- 10025989
- Date Published:
- Journal Name:
- Proc. 21st Annual International Conference on Research in Computational Molecular Biology (RECOMB)
- Volume:
- 21
- Page Range / eLocation ID:
- 402-403
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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Abstract A crucial step in functional genomics is identifying actively translated open reading frames (ORFs) and linking them to biological functions. The challenge lies in identifying short ORFs, as their identification is greatly influenced by data quality and depth. Here, we improved the coverage of super-resolution Ribo-seq in Arabidopsis (Arabidopsis thaliana), revealing uncharacterized translation events for nuclear, chloroplastic, and mitochondrial genes. Assisted by a transcriptome assembly, we identified 7,751 unconventional translation events, comprising 6,996 upstream ORFs (uORFs) and 209 downstream ORFs on annotated protein-coding genes, as well as 546 ORFs in presumed non-coding RNAs. Proteomics data confirmed the production of stable proteins from some of these unannotated translation events. We present evidence of active translation from primary transcripts of tasiRNAs (TAS1–4) and microRNAs (pri-MIR163, pri-MIR169), and periodic ribosome stalling supporting co-translational decay. Additionally, we developed a method for identifying extremely short uORFs, including 370 minimum uORFs (AUG-stop), and 2,921 tiny uORFs (2–10 amino acids), and 681 uORFs that overlap with each other. Remarkably, these short uORFs exhibit strong translational repression as do longer uORFs. We also systematically discovered 594 uORFs regulated by alternative splicing, suggesting widespread isoform-specific translational control. Finally, these prevalent uORFs are associated with numerous important pathways. In summary, our improved Arabidopsis translational landscape provides valuable resources to study gene expression regulation.
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In vivo, proteins fold and function in a complex environment subject to many stresses that can modulate a protein’s energy landscape. One aspect of the environment pertinent to protein folding is the ribosome, since proteins have the opportunity to fold while still bound to the ribosome during translation. We use a combination of force and chemical denaturant (chemomechanical unfolding), as well as point mutations, to characterize the folding mechanism of the src SH3 domain both as a stalled ribosome nascent chain and free in solution. Our results indicate that src SH3 folds through the same pathway on and off the ribosome. Molecular simulations also indicate that the ribosome does not affect the folding pathway for this small protein. Taken together, we conclude that the ribosome does not alter the folding mechanism of this small protein. These results, if general, suggest the ribosome may exert a bigger influence on the folding of multidomain proteins or protein domains that can partially fold before the entire domain sequence is outside the ribosome exit tunnel.
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Abstract Ribosome engineering is a powerful approach for expanding the catalytic potential of the protein synthesis apparatus. Due to the potential detriment the properties of the engineered ribosome may have on the cell, the designer ribosome needs to be functionally isolated from the translation machinery synthesizing cellular proteins. One solution to this problem was offered by Ribo-T, an engineered ribosome with tethered subunits which, while producing a desired protein, could be excluded from general translation. Here, we provide a conceptually different design of a cell with two orthogonal protein synthesis systems, where Ribo-T produces the proteome, while the dissociable ribosome is committed to the translation of a specific mRNA. The utility of this system is illustrated by generating a comprehensive collection of mutants with alterations at every rRNA nucleotide of the peptidyl transferase center and isolating gain-of-function variants that enable the ribosome to overcome the translation termination blockage imposed by an arrest peptide.
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Abstract Elongation factor Tu (EF‐Tu) is a three‐domain protein that is responsible for delivering aminoacyl‐tRNA (aa‐tRNA) molecules to the ribosome. During the delivery process, EF‐Tu undergoes a large‐scale (
~50 Å) conformational transition that results in rearrangement of domain I, relative to the II/III superdomain. Despite the central role of EF‐Tu during protein synthesis, little is known about the structural and energetic properties of this reordering process. To study the physical‐chemical properties of domain motion, we constructed a multi‐basin structure‐based (i.e., Gō‐like) model, with which we have simulated hundreds of spontaneous conformational rearrangements. By analyzing the statistical properties of these events, we show that EF‐Tu is likely to adopt a disordered intermediate ensemble during this transition. We further show that this disordered intermediate will favor a specific sequence of conformational substeps when bound to the ribosome, and the disordered ensemble can influence the kinetics of the incoming aa‐tRNA molecule. Overall, this study highlights the dynamic nature of EF‐Tu by revealing a relationship between conformational disorder and biological function.
- Free Publicly Accessible Full Text
-
Accepted Manuscript1.0
- Conference Paper:
- https://doi.org/10.1007/978-3-319-56970-3
