Evidence for involvement of trans-acting factors in selection of the AUG start codon during eukaryotic translational initiation.

The molecular mechanism with which an appropriate AUG codon is selected as the start site for translational initiation by eukaryotic ribosomes is not known. By using a cell-free translation system, small RNA molecules containing single AUG codons, surrounded by various nucleotide sequences, were tested for their abilities to interfere with the translation of a reporter mRNA. RNAs containing the AUG in an ACCAUGG context (Kozak consensus sequence) were able to inhibit translation of the reporter mRNA. In contrast, RNAs containing the AUG in a less favorable context for start site selection (for example, CAGAUGG) had no effect on the translation of the reporter mRNA. The effect mediated by the ACCAUGC-containing RNAs was not due to sequestration of ribosomal subunits or to particular structural features in these RNAs. To identify potential trans-acting factors that might be preferentially bound by ACCAUGG-containing RNAs, ACCAUGG- and CAGAUGC-containing RNAs with a single 4-thiouridine residue at the AUG were incubated with partially fractionated extracts, and AUG-binding proteins were identified after irradiation of the complexes with UV light and subsequent analysis by gel electrophoresis. The analysis (of such complexes in competition experiments revealed that proteins, approximately 50 and 100 kDa in size, were found to bind directly at the AUG codon embedded in the ACCAUGG motif. One of these proteins has been identified as the La autoantigen. These findings indicate that trans-acting factors may play a role in AUG start site selection during translational initiation.

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Borf: Improved ORF prediction in de-novo assembled transcriptome annotation

10.1101/2021.04.12.439551 ◽

2021 ◽

Author(s):

Beth Signal ◽

Tim Kahlke

Keyword(s):

Site Selection ◽

False Positive ◽

De Novo ◽

Start Codon ◽

Antisense Strand ◽

Upstream Sequence ◽

Start Site ◽

Rna Seq ◽

Sense Strand ◽

The Impact

ABSTRACTORF prediction in de-novo assembled transcriptomes is a critical step for RNA-Seq analysis and transcriptome annotation. However, current approaches do not appropriately account for factors such as strand-specificity and incompletely assembled transcripts. Strand-specific RNA-Seq libraries should produce assembled transcripts in the correct orientation, and therefore ORFs should only be annotated on the sense strand. Additionally, start site selection is more complex than appreciated as sequences upstream of the first start codon need to be correctly annotated as 5’ UTR in completely assembled transcripts, or part of the main ORF in incomplete transcripts. Both of these factors influence the accurate annotation of ORFs and therefore the transcriptome as a whole. We generated four de-novo transcriptome assemblies of well annotated species as a gold-standard dataset to test the impact strand specificity and start site selection have on ORF prediction in real data. Our results show that prediction of ORFs on the antisense strand in data from stranded RNA libraries results in false-positive ORFs with no or very low similarity to known proteins. In addition, we found that up to 23% of assembled transcripts had no stop codon upstream and in-frame of the first start codon, instead comprising a sequence of upstream codons. We found the optimal length cutoff of these upstream sequences to accurately classify these transcripts as either complete (upstream sequence is 5’ UTR) or 5’ incomplete (transcript is incompletely assembled and upstream sequence is part of the ORF). Here, we present Borf, the better ORF finder, specifically designed to minimise false-positive ORF prediction in stranded RNA-Seq data and improve annotation of ORF start-site prediction accuracy. Borf is written in Python3 and freely available at https://github.com/betsig/borf.

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Poly-3-Hydroxybutyrate Degradation in Rhizobium (Sinorhizobium) meliloti: Isolation and Characterization of a Gene Encoding 3-Hydroxybutyrate Dehydrogenase

Journal of Bacteriology ◽

10.1128/jb.181.3.849-857.1999 ◽

1999 ◽

Vol 181 (3) ◽

pp. 849-857 ◽

Cited By ~ 50

Author(s):

P. Aneja ◽

T. C. Charles

Keyword(s):

Carbon Source ◽

Sole Carbon Source ◽

Sinorhizobium Meliloti ◽

Transcriptional Start Site ◽

Consensus Sequence ◽

Start Codon ◽

Start Site ◽

Gene Encoding ◽

Reading Frame ◽

Isolation And Characterization

ABSTRACT We have cloned and sequenced the 3-hydroxybutyrate dehydrogenase-encoding gene (bdhA) from Rhizobium (Sinorhizobium) meliloti. The gene has an open reading frame of 777 bp that encodes a polypeptide of 258 amino acid residues (molecular weight 27,177, pI 6.07). The R. meliloti Bdh protein exhibits features common to members of the short-chain alcohol dehydrogenase superfamily. bdhA is the first gene transcribed in an operon that also includes xdhA, encoding xanthine oxidase/dehydrogenase. Transcriptional start site analysis by primer extension identified two transcription starts. S1, a minor start site, was located 46 to 47 nucleotides upstream of the predicted ATG start codon, while S2, the major start site, was mapped 148 nucleotides from the start codon. Analysis of the sequence immediately upstream of either S1 or S2 failed to reveal the presence of any known consensus promoter sequences. Although a ς54 consensus sequence was identified in the region between S1 and S2, a corresponding transcript was not detected, and a rpoN mutant of R. meliloti was able to utilize 3-hydroxybutyrate as a sole carbon source. The R. meliloti bdhA gene is able to confer uponEscherichia coli the ability to utilize 3-hydroxybutyrate as a sole carbon source. An R. meliloti bdhA mutant accumulates poly-3-hydroxybutyrate to the same extent as the wild type and shows no symbiotic defects. Studies with a strain carrying alacZ transcriptional fusion to bdhAdemonstrated that gene expression is growth phase associated.

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ABC50 mutants modify translation start codon selection

Biochemical Journal ◽

10.1042/bj20141453 ◽

2015 ◽

Vol 467 (2) ◽

pp. 217-229 ◽

Cited By ~ 16

Author(s):

Joanna D. Stewart ◽

Joanne L. Cowan ◽

Lisa S. Perry ◽

Mark J. Coldwell ◽

Christopher G. Proud

Keyword(s):

Site Selection ◽

Initiation Factor ◽

Start Codon ◽

Initiation Codon ◽

Start Site ◽

Key Factors ◽

Initiation Factors ◽

Translation Start ◽

Initiation Factor 2 ◽

Codon Selection

ATP-binding cassette 50 (ABC50; also known as ABCF1) binds to eukaryotic initiation factor 2 (eIF2) and is required for efficient translation initiation. An essential step of this process is accurate recognition and selection of the initiation codon. It is widely accepted that the presence and movement of eIF1, eIF1A and eIF5 are key factors in modulating the stringency of start-site selection, which normally requires an AUG codon in an appropriate sequence context. In the present study, we show that expression of ABC50 mutants, which cannot hydrolyse ATP, decreases general translation and relaxes the discrimination against the use of non-AUG codons at translation start sites. These mutants do not appear to alter the association of key initiation factors to 40S subunits. The stringency of start-site selection can be restored through overexpression of eIF1, consistent with the role of that factor in enhancing stringency. The present study indicates that interfering with the function of ABC50 influences the accuracy of initiation codon selection.

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Quality control of 40S ribosome head assembly ensures scanning competence

The Journal of Cell Biology ◽

10.1083/jcb.202004161 ◽

2020 ◽

Vol 219 (11) ◽

Author(s):

Haina Huang ◽

Homa Ghalei ◽

Katrin Karbstein

Keyword(s):

Quality Control ◽

Conformational Changes ◽

Translation Initiation ◽

Site Selection ◽

Start Codon ◽

Start Site ◽

Genetic Analyses ◽

Head Assembly ◽

Control Step ◽

Assembly Intermediate

During translation initiation, 40S ribosomes scan the mRNA until they encounter the start codon, where conformational changes produce a translation-competent 80S complex. Destabilizing the scanning complex results in misinitiation at non-AUG codons, demonstrating its importance for fidelity. Here, we use a combination of biochemical and genetic analyses to demonstrate that the ability of the nascent subunit to adopt the scanning complex is tested during assembly via structural mimicry. Specifically, formation of the 80S-like assembly intermediate, which structurally resembles scanning complexes, requires the correct folding of two rRNA elements in the subunit head and the proper positioning of the universally conserved head proteins Rps3, Rps15, Rps20, and Rps29. rRNA misfolding impairs the formation of 80S-like ribosomes, and bypass of individual checkpoints using cancer-associated mutations produces ribosomes defective in accurate start-site selection. Thus, the formation of 80S-like assembly intermediates is a quality control step that ensures scanning competence of the nascent subunit.

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Faculty Opinions recommendation of Mechanism of start site selection by RNA polymerase II: interplay between TFIIB and Ssl2/XPB helicase subunit of TFIIH.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.13968976.15426085 ◽

2012 ◽

Author(s):

Daniel Reines

Keyword(s):

Rna Polymerase ◽

Rna Polymerase Ii ◽

Site Selection ◽

Start Site

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Start site selection at the TATA-less carbamoyl-phosphate synthase (glutamine-hydrolyzing)/aspartate carbamoyltransferase/dihydroorotase promoter.

Journal of Biological Chemistry ◽

10.1016/s0021-9258(17)42161-2 ◽

1994 ◽

Vol 269 (3) ◽

pp. 2252-2257

Author(s):

R. Kollmar ◽

K.A. Sukow ◽

S.K. Sponagle ◽

P.J. Farnham

Keyword(s):

Site Selection ◽

Start Site ◽

Carbamoyl Phosphate ◽

Aspartate Carbamoyltransferase ◽

Phosphate Synthase

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Communication between Eukaryotic Translation Initiation Factors 5 and 1A within the Ribosomal Pre-initiation Complex Plays a Role in Start Site Selection

Journal of Molecular Biology ◽

10.1016/j.jmb.2005.11.083 ◽

2006 ◽

Vol 356 (3) ◽

pp. 724-737 ◽

Cited By ~ 57

Author(s):

David Maag ◽

Mikkel A. Algire ◽

Jon R. Lorsch

Keyword(s):

Translation Initiation ◽

Site Selection ◽

Start Site ◽

Initiation Complex ◽

Eukaryotic Translation ◽

Initiation Factors ◽

Translation Initiation Factors ◽

Eukaryotic Translation Initiation ◽

Eukaryotic Translation Initiation Factors

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A Mutation in Paramecium tetraurelia Reveals Functional and Structural Features of Developmentally Excised DNA Elements

Genetics ◽

10.1093/genetics/148.1.139 ◽

1998 ◽

Vol 148 (1) ◽

pp. 139-149 ◽

Cited By ~ 4

Author(s):

Kimberly M Mayer ◽

Kazuyuki Mikami ◽

James D Forney

Keyword(s):

Mutant Cell ◽

Consensus Sequence ◽

Surface Protein ◽

Structural Features ◽

Inverted Terminal Repeat ◽

Paramecium Tetraurelia ◽

Coding Region ◽

Conserved Sequence ◽

Variable Surface ◽

Dna Elements

Abstract The excision of internal eliminated sequences (IESs) from the germline micronuclear DNA occurs during the differentiation of a new macronuclear genome in ciliated protozoa. In Paramecium, IESs are generally short (28–882 bp), AT rich DNA elements that show few conserved sequence features with the exception of an inverted-terminal-repeat consensus sequence that has similarity to the ends of mariner/Tc1 transposons (Klobutcher and Herrick 1995). We have isolated and analyzed a mutant cell line that cannot excise a 370-bp IESs (IES2591) from the coding region of the 51A variable surface protein gene. A single micronuclear C to T transition within the consensus sequence prevents excision. The inability to excise IES2591 has revealed a 28-bp IES inside the larger IES, suggesting that reiterative integration of these elements can occur. Together, the consensus sequence mutation and the evidence for reiterative integration support the theory that Paramecium IESs evolved from transposable elements. Unlike a previously studied Paramecium IES, the presence of this IES in the macronucleus does not completely inhibit excision of its wild-type micronuclear copy through multiple sexual generations.

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