“…The most common start codons for known Escherichia coli genes are AUG (83% of genes), GUG (14%) and UUG (3%) (2–4). Similar percentages can be found throughout the bacterial domain (5). …”
Our understanding of translation underpins our capacity to engineer living systems. The canonical start codon (AUG) and a few near-cognates (GUG, UUG) are considered as the ‘start codons’ for translation initiation in Escherichia coli. Translation is typically not thought to initiate from the 61 remaining codons. Here, we quantified translation initiation of green fluorescent protein and nanoluciferase in E. coli from all 64 triplet codons and across a range of DNA copy number. We detected initiation of protein synthesis above measurement background for 47 codons. Translation from non-canonical start codons ranged from 0.007 to 3% relative to translation from AUG. Translation from 17 non-AUG codons exceeded the highest reported rates of non-cognate codon recognition. Translation initiation from non-canonical start codons may contribute to the synthesis of peptides in both natural and synthetic biological systems.
“…The most common start codons for known Escherichia coli genes are AUG (83% of genes), GUG (14%) and UUG (3%) (2–4). Similar percentages can be found throughout the bacterial domain (5). …”
Our understanding of translation underpins our capacity to engineer living systems. The canonical start codon (AUG) and a few near-cognates (GUG, UUG) are considered as the ‘start codons’ for translation initiation in Escherichia coli. Translation is typically not thought to initiate from the 61 remaining codons. Here, we quantified translation initiation of green fluorescent protein and nanoluciferase in E. coli from all 64 triplet codons and across a range of DNA copy number. We detected initiation of protein synthesis above measurement background for 47 codons. Translation from non-canonical start codons ranged from 0.007 to 3% relative to translation from AUG. Translation from 17 non-AUG codons exceeded the highest reported rates of non-cognate codon recognition. Translation initiation from non-canonical start codons may contribute to the synthesis of peptides in both natural and synthetic biological systems.
“…Protein sequencing revealed the 49 kDa protein to be X SMOC-1, whereas the 24 kDa protein was a partial X SMOC-1 sequence beginning at V235. The base sequence (GTG) was consistent with an alternative start codon (Villegas and Kropinski, 2008); when changed to GTA by site-directed mutagenesis only the expected 49 kDa product was produced (Figure 2A). All subsequent X SMOC-1 and X SMOC-1∆EC constructs contained GTA at V235.10.7554/eLife.17935.006Figure 2.Expression and refolding of recombinant mature X SMOC-1, X SMOC-1∆EC, and X SMOC-1EC.( A ) Coomassie stained SDS-PAGE of wild type (V235-GTG) and silent mutant (V235-GTA) recombinant mature X SMOC-1 following size exclusion chromatography (SEC).…”
Section: Resultsmentioning
confidence: 88%
“…When pent was expressed in its normal location during wing development, using the Gal4/UAS system, the mutant phenotype was rescued completely, demonstrating that the pent construct had full biological activity (Figure 1—figure supplement 1). Initial injections of pent mRNA into Xenopus embryos produced no apparent effects (not shown); however overexpression of a synthetic pent mRNA ( co-pent ) optimized for codon usage and translation efficiency in Xenopus (Villegas and Kropinski, 2008) (Figure 1—figure supplement 2) produced a dorsalized phenotype indistinguishable from that observed following overexpression of Xenopus SMOC-1 ( X SMOC-1) (Figure 1B). The ability of Pent to inhibit BMP signaling downstream of the BMP receptor was analyzed in Xenopus ectodermal explants (animal caps) following co-injection of mRNAs for co-pent and constitutively active BMP receptor1B (caBMPR1B); the caBMPR1B, containing an intracellular activating mutation (Q203D), promotes phosphorylation of Smad 1/5/8 and subsequent BMP signaling events independent of ligand binding (Zou et al, 1997).…”
The matricellular protein SMOC (Secreted Modular Calcium binding protein) is conserved phylogenetically from vertebrates to arthropods. We showed previously that SMOC inhibits bone morphogenetic protein (BMP) signaling downstream of its receptor via activation of mitogen-activated protein kinase (MAPK) signaling. In contrast, the most prominent effect of the Drosophila orthologue, pentagone (pent), is expanding the range of BMP signaling during wing patterning. Using SMOC deletion constructs we found that SMOC-∆EC, lacking the extracellular calcium binding (EC) domain, inhibited BMP2 signaling, whereas SMOC-EC (EC domain only) enhanced BMP2 signaling. The SMOC-EC domain bound HSPGs with a similar affinity to BMP2 and could expand the range of BMP signaling in an in vitro assay by competition for HSPG-binding. Together with data from studies in vivo we propose a model to explain how these two activities contribute to the function of Pent in Drosophila wing development and SMOC in mammalian joint formation.DOI:
http://dx.doi.org/10.7554/eLife.17935.001
“…The most frequent initiation codon in B. methanolicus MGA3 is ATG (75.5%), a frequency typical for 620 complete bacterial genomes (80.1%; [54]). As in B. subtilis [55], the generally uncommon initiation codon TTG (13.9%) was more common than GTG (10.6%) in B. methanolicus MGA3.…”
BackgroundBacillus methanolicus MGA3 is a thermophilic, facultative ribulose monophosphate (RuMP) cycle methylotroph. Together with its ability to produce high yields of amino acids, the relevance of this microorganism as a promising candidate for biotechnological applications is evident. The B. methanolicus MGA3 genome consists of a 3,337,035 nucleotides (nt) circular chromosome, the 19,174 nt plasmid pBM19 and the 68,999 nt plasmid pBM69. 3,218 protein-coding regions were annotated on the chromosome, 22 on pBM19 and 82 on pBM69. In the present study, the RNA-seq approach was used to comprehensively investigate the transcriptome of B. methanolicus MGA3 in order to improve the genome annotation, identify novel transcripts, analyze conserved sequence motifs involved in gene expression and reveal operon structures. For this aim, two different cDNA library preparation methods were applied: one which allows characterization of the whole transcriptome and another which includes enrichment of primary transcript 5′-ends.ResultsAnalysis of the primary transcriptome data enabled the detection of 2,167 putative transcription start sites (TSSs) which were categorized into 1,642 TSSs located in the upstream region (5′-UTR) of known protein-coding genes and 525 TSSs of novel antisense, intragenic, or intergenic transcripts. Firstly, 14 wrongly annotated translation start sites (TLSs) were corrected based on primary transcriptome data. Further investigation of the identified 5′-UTRs resulted in the detailed characterization of their length distribution and the detection of 75 hitherto unknown cis-regulatory RNA elements. Moreover, the exact TSSs positions were utilized to define conserved sequence motifs for translation start sites, ribosome binding sites and promoters in B. methanolicus MGA3. Based on the whole transcriptome data set, novel transcripts, operon structures and mRNA abundances were determined. The analysis of the operon structures revealed that almost half of the genes are transcribed monocistronically (940), whereas 1,164 genes are organized in 381 operons. Several of the genes related to methylotrophy had highly abundant transcripts.ConclusionThe extensive insights into the transcriptional landscape of B. methanolicus MGA3, gained in this study, represent a valuable foundation for further comparative quantitative transcriptome analyses and possibly also for the development of molecular biology tools which at present are very limited for this organism.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-015-1239-4) contains supplementary material, which is available to authorized users.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
