Global analysis of protein synthesis in Flavobacterium johnsoniae reveals the use of Kozak-like sequences in diverse bacteria

Abstract In all cells, initiation of translation is tuned by intrinsic features of the mRNA. Here, we analyze translation in Flavobacterium johnsoniae, a representative of the Bacteroidetes. Members of this phylum naturally lack Shine–Dalgarno (SD) sequences in their mRNA, and yet their ribosomes retain the conserved anti-SD sequence. Translation initiation is tuned by mRNA secondary structure and by the identities of several key nucleotides upstream of the start codon. Positive determinants include adenine at position –3, reminiscent of the Kozak sequence of Eukarya. Comparative analysis of Escherichia coli reveals use of the same Kozak-like sequence to enhance initiation, suggesting an ancient and widespread mechanism. Elimination of contacts between A-3 and the conserved β-hairpin of ribosomal protein uS7 fails to diminish the contribution of A-3 to initiation, suggesting an indirect mode of recognition. Also, we find that, in the Bacteroidetes, the trinucleotide AUG is underrepresented in the vicinity of the start codon, which presumably helps compensate for the absence of SD sequences in these organisms.

Download Full-text

Contributions of the N- and C-Terminal Domains of Initiation Factor 3 to Its Functions in the Fidelity of Initiation and Antiassociation of the Ribosomal Subunits

Journal of Bacteriology ◽

10.1128/jb.00051-17 ◽

2017 ◽

Vol 199 (11) ◽

Cited By ~ 7

Author(s):

Shreya Ahana Ayyub ◽

Divya Dobriyal ◽

Umesh Varshney

Keyword(s):

Escherichia Coli ◽

Protein Synthesis ◽

Bacterial Growth ◽

Translation Initiation ◽

Ribosomal Subunit ◽

Initiation Factor ◽

Start Codon ◽

Content Type ◽

E Coli

ABSTRACT Initiation factor 3 (IF3) is one of the three conserved prokaryotic translation initiation factors essential for protein synthesis and cellular survival. Bacterial IF3 is composed of a conserved architecture of globular N- and C-terminal domains (NTD and CTD) joined by a linker region. IF3 is a ribosome antiassociation factor which also modulates selection of start codon and initiator tRNA. All the functions of IF3 have been attributed to its CTD by in vitro studies. However, the in vivo relevance of these findings has not been investigated. By generating complete and partial IF3 (infC) knockouts in Escherichia coli and by complementation analyses using various deletion constructs, we show that while the CTD is essential for E. coli survival, the NTD is not. Polysome profiles reaffirm that CTD alone can bind to the 30S ribosomal subunit and carry out the ribosome antiassociation function. Importantly, in the absence of the NTD, bacterial growth is compromised, indicating a role for the NTD in the fitness of cellular growth. Using reporter assays for in vivo initiation, we show that the NTD plays a crucial role in the fidelity function of IF3 by avoiding (i) initiation from non-AUG codons and (ii) initiation by initiator tRNAs lacking the three highly conserved consecutive GC pairs (in the anticodon stem) known to function in concert with IF3. IMPORTANCE Initiation factor 3 regulates the fidelity of eubacterial translation initiation by ensuring the formation of an initiation complex with an mRNA bearing a canonical start codon and with an initiator tRNA at the ribosomal P site. Additionally, IF3 prevents premature association of the 50S ribosomal subunit with the 30S preinitiation complex. The significance of our work in Escherichia coli is in demonstrating that while the C-terminal domain alone sustains E. coli for its growth, the N-terminal domain adds to the fidelity of initiation of protein synthesis and to the fitness of the bacterial growth.

Download Full-text

Measurements of translation initiation from all 64 codons inE. coli

10.1101/063800 ◽

2016 ◽

Author(s):

Ariel Hecht ◽

Jeff Glasgow ◽

Paul R. Jaschke ◽

Lukmaan Bawazer ◽

Matthew S. Munson ◽

...

Keyword(s):

Escherichia Coli ◽

Protein Synthesis ◽

Molecular Biology ◽

Translation Initiation ◽

Biological Systems ◽

Start Codon ◽

Living Matter ◽

E Coli ◽

Natural And Synthetic

ABSTRACTOur understanding of translation is one cornerstone of molecular biology that underpins our capacity to engineer living matter. The canonical start codon (AUG) and a few near-cognates (GUG, UUG) are typically considered as the “start codons” for translation initiation inEscherichia coli(E. coli). Translation is typically not thought to initiate from the 61 remaining codons. Here, we systematically quantified translation initiation inE. colifrom all 64 triplet codons. We detected protein synthesis above background initiating from at least 46 codons. Translation initiated from these non-canonical start codons at levels ranging from 0.01% to 2% relative to AUG. Translation initiation from non-canonical start codons may contribute to the synthesis of peptides in both natural and synthetic biological systems

Download Full-text

Caspase-mediated cleavage of eukaryotic translation initiation factor subunit 2α

Biochemical Journal ◽

10.1042/bj3420065 ◽

1999 ◽

Vol 342 (1) ◽

pp. 65-70 ◽

Cited By ~ 31

Author(s):

Shinya SATOH ◽

Makoto HIJIKATA ◽

Hiroshi HANDA ◽

Kunitada SHIMOTOHNO

Keyword(s):

Protein Synthesis ◽

Translation Initiation ◽

Caspase 3 ◽

Translation Initiation Factor ◽

Initiation Factor ◽

Eukaryotic Translation Initiation Factor ◽

Α Subunit ◽

Eukaryotic Translation ◽

Initiation Of Translation ◽

Eukaryotic Translation Initiation

Eukaryotic translation initiation factor 2α (eIF-2α), a target molecule of the interferon-inducible double-stranded-RNA-dependent protein kinase (PKR), was cleaved in apoptotic Saos-2 cells on treatment with poly(I)˙poly(C) or tumour necrosis factor α. This cleavage occurred with a time course similar to that of poly(ADP-ribose) polymerase, a well-known caspase substrate. In addition, eIF-2α was cleaved by recombinant active caspase-3 in vitro. By site-directed mutagenesis, the cleavage site was mapped to an Ala-Glu-Val-Asp300 ↓ Gly301 sequence located in the C-terminal portion of eIF-2α. PKR phosphorylates eIF-2α on Ser51, resulting in the suppression of protein synthesis. PKR-mediated translational suppression was repressed when the C-terminally cleaved product of eIF-2α was overexpressed in Saos-2 cells, even though PKR can phosphorylate this cleaved product. These results suggest that caspase-3 or related protease(s) can modulate the efficiency of protein synthesis by cleaving the α subunit of eIF-2, a key component in the initiation of translation.

Download Full-text

Bacteria differently regulate mRNA abundance to specifically respond to various stresses

Philosophical Transactions of The Royal Society A Mathematical Physical and Engineering Sciences ◽

10.1098/rsta.2015.0069 ◽

2016 ◽

Vol 374 (2063) ◽

pp. 20150069 ◽

Cited By ~ 39

Author(s):

Alexander Bartholomäus ◽

Ivan Fedyunin ◽

Peter Feist ◽

Celine Sin ◽

Gong Zhang ◽

...

Keyword(s):

Escherichia Coli ◽

Stress Response ◽

Global Analysis ◽

Start Codon ◽

Specific Response ◽

Copy Numbers ◽

Genome Wide ◽

A Genome ◽

Temperature Upshift ◽

Global Reduction

Environmental stress is detrimental to cell viability and requires an adequate reprogramming of cellular activities to maximize cell survival. We present a global analysis of the response of Escherichia coli to acute heat and osmotic stress. We combine deep sequencing of total mRNA and ribosome-protected fragments to provide a genome-wide map of the stress response at transcriptional and translational levels. For each type of stress, we observe a unique subset of genes that shape the stress-specific response. Upon temperature upshift, mRNAs with reduced folding stability up- and downstream of the start codon, and thus with more accessible initiation regions, are translationally favoured. Conversely, osmotic upshift causes a global reduction of highly translated transcripts with high copy numbers, allowing reallocation of translation resources to not degraded and newly synthesized mRNAs.

Download Full-text

Eukaryotic aspects of translation initiation brought into focus

Philosophical Transactions of the Royal Society B Biological Sciences ◽

10.1098/rstb.2016.0186 ◽

2017 ◽

Vol 372 (1716) ◽

pp. 20160186 ◽

Cited By ~ 19

Author(s):

Christopher H. S. Aylett ◽

Nenad Ban

Keyword(s):

Translation Initiation ◽

Structural Information ◽

Start Codon ◽

Translation Regulation ◽

Initiation Factors ◽

Codon Recognition ◽

Initiation Of Translation ◽

Control Translation ◽

Additional Level ◽

Subunit Joining

In all organisms, mRNA-directed protein synthesis is catalysed by ribosomes. Although the basic aspects of translation are preserved in all kingdoms of life, important differences are found in the process of translation initiation, which is rate-limiting and the most important step for translation regulation. While great strides had been taken towards a complete structural understanding of the initiation of translation in eubacteria, our understanding of the eukaryotic process, which includes numerous eukaryotic-specific initiation factors, was until recently limited owing to a lack of structural information. In this review, we discuss recent results in the field that provide an increasingly complete molecular description of the eukaryotic initiation process. The structural snapshots obtained using a range of methods now provide insights into the architecture of the initiation complex, start-codon recognition by the initiator tRNA and the process of subunit joining. Future advances will require both higher-resolution insights into previously characterized complexes and mapping of initiation factors that control translation on an additional level by interacting only peripherally or transiently with ribosomal subunits. This article is part of the themed issue ‘Perspectives on the ribosome’.

Download Full-text

Kinetic modeling predicts a stimulatory role for ribosome collisions at elongation stall sites in bacteria

eLife ◽

10.7554/elife.23629 ◽

2017 ◽

Vol 6 ◽

Cited By ~ 18

Author(s):

Michael A Ferrin ◽

Arvind R Subramaniam

Keyword(s):

Escherichia Coli ◽

Quality Control ◽

Protein Synthesis ◽

Translation Initiation ◽

Computational Analysis ◽

Fine Tuning ◽

Initiation Rate ◽

Kinetic Rate ◽

Ribosome Stalling ◽

Traffic Jams

Ribosome stalling on mRNAs can decrease protein expression. To decipher ribosome kinetics at stall sites, we induced ribosome stalling at specific codons by starving the bacterium Escherichia coli for the cognate amino acid. We measured protein synthesis rates from a reporter library of over 100 variants that encoded systematic perturbations of translation initiation rate, the number of stall sites, and the distance between stall sites. Our measurements are quantitatively inconsistent with two widely-used kinetic models for stalled ribosomes: ribosome traffic jams that block initiation, and abortive (premature) termination of stalled ribosomes. Rather, our measurements support a model in which collision with a trailing ribosome causes abortive termination of the stalled ribosome. In our computational analysis, ribosome collisions selectively stimulate abortive termination without fine-tuning of kinetic rate parameters at ribosome stall sites. We propose that ribosome collisions serve as a robust timer for translational quality control pathways to recognize stalled ribosomes.

Download Full-text

117 TRANSLATION INITIATION IS ESSENTIALLY MODIFIED IN THE PORCINE ENDOMETRIAL TISSUE DURING EARLY PREGNANCY

Reproduction Fertility and Development ◽

10.1071/rdv25n1ab117 ◽

2013 ◽

Vol 25 (1) ◽

pp. 206

Author(s):

K. Wollenhaupt ◽

K. P. Brüssow ◽

W. Tomek

Keyword(s):

Protein Synthesis ◽

Translation Initiation ◽

Early Pregnancy ◽

Mapk Pathway ◽

Binding Protein ◽

Sequence Motif ◽

Initiation Of Translation ◽

Essential Components ◽

Cap Binding Complex

Protein synthesis is a major determinate in cell growth and differentiation. Here, we investigated the porcine uterine tissue during early pregnancy. The objective was the analysis of the initiation of translation, which is believed to be the rate-limiting process of protein synthesis. Performing Western blot screening during implantation and placentation, in vitro protease assays and mass spectrometry, we revealed that essential components of the cap-binding complex eIF4F and its regulators are targets of specific proteolytically processing. This proteolytical activity shifts from the implantation site to the peri-placental area later in development. The results show that the cap-binding protein eIF4E is N-terminally truncated which results in lower binding to the repressors 4E-BP1 and 4E-BP2. On the other hand, 4E-BP1 is mainly degraded, whereas 4E-BP2 emerges as N-terminally truncated stable fragments. It is likely that these fragments have lost an essential sequence motif that enables the phosphorylation and inactivation of the repressor. Furthermore, the Akt/mTor signaling cascade (which phosphorylates 4E-BP1 and 2) but not the MAPK pathway (which phosphorylated eIF4E and together with Akt/mTor hierarchically 4E-BP1 and 2) was inactivated. This results in long-term translation repression of eIF4E-sensitive and 4E-BP2-dependent mRNAs. However, other translation initiation factors like eIF4A, eIF2α, and the poly(A)-binding protein PABP are not modified. In conclusion, we suggest that the observed specific modification of the mRNA-cap-binding complex in the porcine endometrium during early pregnancy is essential to regulate gene expression to establish the noninvasive implantation and to sustain the ongoing gravidity.

Download Full-text

On translational control by ribosome speed inS. cerevisiae

10.1101/513705 ◽

2019 ◽

Author(s):

Eleanna Kazana ◽

Tobias von der Haar

Keyword(s):

Protein Synthesis ◽

Translation Initiation ◽

Translational Control ◽

Start Codon ◽

Equal Proportion ◽

Translation Elongation ◽

Show Evidence ◽

Genome Wide Data ◽

Yeast Genes ◽

Elongation Control

AbstractIn addition to the widespread and well documented control of protein synthesis by translation initiation, recent evidence suggests that translation elongation can also control protein synthesis rates. One of the proposed mechanisms leading to elongation control is the interference of slow ribosome movement around the start codon with efficient translation initiation. Here we estimate the frequency with which this mode of control occurs in baker’s yeast growing in rich medium. Genome-wide data reveal that transcripts from around 20% of yeast genes show evidence of queueing ribosomes, which may be indicative of translation elongation control. Moreover, this subset of transcripts is sensitive to distinct regulatory signals compared to initiation-controlled mRNAs, and such distinct regulation occurs for example during the response to osmotic stress.Notethe previous version 2 of this preprint contained a Decision-Tree based analysis where we attempted to relate mRNA features to the presence or absence of queueing ribosome peaks. Since releasing that version, we performed additional controls for this analysis which strongly sugest that its results are random and should be ignored. Specifically, both the overall predictability and the importance of individual features is very similar for a real dataset where genes are labelled as containing a second SSU peak or not, and for a simulated control dataset containing an equal proportion of randomly labelled genes. We have removed this figure from the current version. The analysis together with the additional control remains accessible on the accompanying Github repository (github.com/tobiasvonderhaar/ribosomespeedcontrol) in the file “Figure 3 Obsolete (Classification).ipynb”.

Download Full-text