scholarly journals MOD5 translation initiation sites determine N6-isopentenyladenosine modification of mitochondrial and cytoplasmic tRNA.

1991 ◽  
Vol 11 (5) ◽  
pp. 2382-2390 ◽  
Author(s):  
E C Gillman ◽  
L B Slusher ◽  
N C Martin ◽  
A K Hopper
Keyword(s):  
Trna Modification ◽  
Cellular Compartment ◽  
Reading Frame ◽  
Transcription Start Sites ◽  
Amino Terminal ◽  
Translation Start ◽  
Long Form ◽  
Cellular Compartments ◽  
Selection Of

MOD5 is one of several genes that code for enzymes found in mitochondria and another cellular compartment. Like other such genes, it contains two in-frame ATGs that could be used to produce two proteins, differing from each other by an amino-terminal extension. Certain other genes produce heterogeneous mRNAs with some 5' ends falling upstream of the longest open reading frame and some 5' ends falling between the first and second ATGs. In these cases, selection of transcription start sites appears to play a significant role in translation start site selection. MOD5, in contrast, produces mRNAs with 5' ends that all fall upstream of both ATGs. To determine how MOD5 encodes isozymes that are located in different cellular compartments and to determine whether they differ in structure, we constructed MOD5 and MOD5-COXIV fusions with mutations of the first, second, or both ATGs. The effect of these alterations on protein production, tRNA modification, and cellular location was assessed. Both the first and second ATGs are used to produce MOD5 protein in vivo, but only the long form of the protein is imported into mitochondria. Thus, the first 11 amino acids present on the amino-terminal extended protein are necessary for mitochondrial import. Surprisingly, this extension does not promote complete import of the long form of the protein, but rather a functional pool of the extended protein remains in the cytoplasm. The amino-terminal extension is also unusual because it is probably not proteolytically removed upon import and therefore does not constitute part of a mitochondrial presequence.

MOD5 translation initiation sites determine N6-isopentenyladenosine modification of mitochondrial and cytoplasmic tRNA

1991 ◽  
Vol 11 (5) ◽  
pp. 2382-2390
Author(s):  
E C Gillman ◽  
L B Slusher ◽  
N C Martin ◽  
A K Hopper
Keyword(s):  
Trna Modification ◽  
Cellular Compartment ◽  
Reading Frame ◽  
Transcription Start Sites ◽  
Amino Terminal ◽  
Translation Start ◽  
Long Form ◽  
Cellular Compartments ◽  
Selection Of

MOD5 is one of several genes that code for enzymes found in mitochondria and another cellular compartment. Like other such genes, it contains two in-frame ATGs that could be used to produce two proteins, differing from each other by an amino-terminal extension. Certain other genes produce heterogeneous mRNAs with some 5' ends falling upstream of the longest open reading frame and some 5' ends falling between the first and second ATGs. In these cases, selection of transcription start sites appears to play a significant role in translation start site selection. MOD5, in contrast, produces mRNAs with 5' ends that all fall upstream of both ATGs. To determine how MOD5 encodes isozymes that are located in different cellular compartments and to determine whether they differ in structure, we constructed MOD5 and MOD5-COXIV fusions with mutations of the first, second, or both ATGs. The effect of these alterations on protein production, tRNA modification, and cellular location was assessed. Both the first and second ATGs are used to produce MOD5 protein in vivo, but only the long form of the protein is imported into mitochondria. Thus, the first 11 amino acids present on the amino-terminal extended protein are necessary for mitochondrial import. Surprisingly, this extension does not promote complete import of the long form of the protein, but rather a functional pool of the extended protein remains in the cytoplasm. The amino-terminal extension is also unusual because it is probably not proteolytically removed upon import and therefore does not constitute part of a mitochondrial presequence.

DNA sequence and transcript mapping of MOD5: features of the 5' region which suggest two translational starts

1987 ◽  
Vol 7 (1) ◽  
pp. 185-191
Author(s):  
D Najarian ◽  
M E Dihanich ◽  
N C Martin ◽  
A K Hopper
Keyword(s):  
Dna Sequence ◽  
Nuclear Gene ◽  
Open Reading Frame ◽  
Isopentenyl Transferase ◽  
Gene Encoding ◽  
Reading Frame ◽  
Amino Terminal ◽  
Initiation Of Translation ◽  
Cellular Compartments ◽  
Lines Of Evidence

A mutation in the yeast nuclear gene MOD5 drastically reduces the biosynthesis of the modified base isopentenyladenosine in tRNAs located in different cellular compartments: the mitochondria and the nucleus or cytoplasm. Several lines of evidence strongly suggest that MOD5 is the structural gene encoding the tRNA-modifying enzyme delta 2-isopentenyl pyrophosphate:tRNA isopentenyl transferase. DNA sequence analysis of MOD5 reveals an open reading frame of 428 amino acids. A set of mRNAs heterogeneous at both the 5' and 3' termini are transcribed from this gene. Although all of these transcripts initiate upstream of the first AUG codon of the open reading frame, a subset has an extremely short (greater than or equal to 1 base) 5' leader. Furthermore, in positions important for efficient initiation of translation and generally occupied by purines, this first AUG codon is flanked by a U (position -3) and a C (position +4). It is possible that two proteins, one with an amino-terminal extension of basic charge, could be generated from the MOD5 gene via differential translational starts.

scholarly journals Two isoforms of Saccharomyces cerevisiae glutaredoxin 2 are expressed in vivo and localize to different subcellular compartments

2002 ◽  
Vol 364 (3) ◽  
pp. 617-623 ◽  
Author(s):  
José R. PEDRAJAS ◽  
Pablo PORRAS ◽  
Emilia MARTÍNEZ-GALISTEO ◽  
C. Alicia PADILLA ◽  
Antonio MIRANDA-VIZUETE ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Exponential Phase ◽  
Start Codon ◽  
Immunochemical Analysis ◽  
Translation Product ◽  
Differential Processing ◽  
Reading Frame ◽  
Yeast Extract Peptone Dextrose ◽  
Long Form

Glutaredoxin (Grx)2 from Saccharomyces cerevisiae is a member of the two-cysteine (dithiol) subfamily of Grxs involved in the defence against oxidative stress in yeast. Recombinant yeast Grx2p, expressed in Escherichia coli, behaves as a ‘classical’ Grx that efficiently catalyses the reduction of hydroxyethyl disulphide by GSH. Grx2p also catalyses the reduction of GSSG by dihydrolipoamide with even higher efficiency. Western blot analysis of S. cerevisiae crude extracts identifies two isoforms of Grx2p of 15.9 and 11.9kDa respectively. The levels of these two isoforms reach a peak during the exponential phase of growth in normal yeast extract/peptone/dextrose ('YPD') medium, with the long form predominating over the short one. From immunochemical analysis of subcellular fractions, it is shown that both isoforms are present in mitochondria, but only the short one is detected in the cytosolic fraction. On the other hand, only the long form is prominent in microsomes. Mitochondrial isoforms should represent the processed and unprocessed products of an open reading frame (YDR513W), with a putative start codon 99bp upstream of the GRX2 start codon described thus far. These results indicate that GRX2 contains two in-frame start codons, and that translation from the first AUG results in a product that is targeted to mitochondria. The cytosolic form would result either by initiation from the second AUG, or by differential processing of one single translation product.

scholarly journals Engineering gene overlaps to sustain genetic constructs in vivo

2021 ◽  
Vol 17 (10) ◽  
pp. e1009475
Author(s):  
Antoine L. Decrulle ◽  
Antoine Frénoy ◽  
Thomas A. Meiller-Legrand ◽  
Aude Bernheim ◽  
Chantal Lotton ◽  
...  
Keyword(s):  
Essential Gene ◽  
Loss Of Function ◽  
Evolutionary Potential ◽  
Reading Frame ◽  
E Coli ◽  
Overlapping Reading Frames ◽  
The Stability ◽  
Genetic Constructs ◽  
Selection Of

Evolution is often an obstacle to the engineering of stable biological systems due to the selection of mutations inactivating costly gene circuits. Gene overlaps induce important constraints on sequences and their evolution. We show that these constraints can be harnessed to increase the stability of costly genes by purging loss-of-function mutations. We combine computational and synthetic biology approaches to rationally design an overlapping reading frame expressing an essential gene within an existing gene to protect. Our algorithm succeeded in creating overlapping reading frames in 80% of E. coli genes. Experimentally, scoring mutations in both genes of such overlapping construct, we found that a significant fraction of mutations impacting the gene to protect have a deleterious effect on the essential gene. Such an overlap thus protects a costly gene from removal by natural selection by associating the benefit of this removal with a larger or even lethal cost. In our synthetic constructs, the overlap converts many of the possible mutants into evolutionary dead-ends, reducing the evolutionary potential of the system and thus increasing its stability over time.

scholarly journals DNA sequence and transcript mapping of MOD5: features of the 5' region which suggest two translational starts.

1987 ◽  
Vol 7 (1) ◽  
pp. 185-191 ◽  
Author(s):  
D Najarian ◽  
M E Dihanich ◽  
N C Martin ◽  
A K Hopper
Keyword(s):  
Dna Sequence ◽  
Nuclear Gene ◽  
Open Reading Frame ◽  
Isopentenyl Transferase ◽  
Gene Encoding ◽  
Reading Frame ◽  
Amino Terminal ◽  
Initiation Of Translation ◽  
Cellular Compartments ◽  
Lines Of Evidence

A mutation in the yeast nuclear gene MOD5 drastically reduces the biosynthesis of the modified base isopentenyladenosine in tRNAs located in different cellular compartments: the mitochondria and the nucleus or cytoplasm. Several lines of evidence strongly suggest that MOD5 is the structural gene encoding the tRNA-modifying enzyme delta 2-isopentenyl pyrophosphate:tRNA isopentenyl transferase. DNA sequence analysis of MOD5 reveals an open reading frame of 428 amino acids. A set of mRNAs heterogeneous at both the 5' and 3' termini are transcribed from this gene. Although all of these transcripts initiate upstream of the first AUG codon of the open reading frame, a subset has an extremely short (greater than or equal to 1 base) 5' leader. Furthermore, in positions important for efficient initiation of translation and generally occupied by purines, this first AUG codon is flanked by a U (position -3) and a C (position +4). It is possible that two proteins, one with an amino-terminal extension of basic charge, could be generated from the MOD5 gene via differential translational starts.

scholarly journals The Amino-Terminal 100 Residues of the Nitrogen Assimilation Control Protein (NAC) Encode All Known Properties of NAC from Klebsiella aerogenes and Escherichia coli

1999 ◽  
Vol 181 (3) ◽  
pp. 934-940 ◽  
Author(s):  
Wilson B. Muse ◽  
Robert A. Bender
Keyword(s):  
Escherichia Coli ◽  
Amino Acids ◽  
Nitrogen Assimilation ◽  
Factor Xa ◽  
Klebsiella Aerogenes ◽  
Reading Frame ◽  
Amino Terminal ◽  
Control Protein

ABSTRACT The nitrogen assimilation control protein (NAC) fromKlebsiella aerogenes or Escherichia coli(NACK or NACE, respectively) is a transcriptional regulator that is both necessary and sufficient to activate transcription of the histidine utilization (hut) operon and to repress transcription of the glutamate dehydrogenase (gdh) operon in K. aerogenes. Truncated NAC polypeptides, generated by the introduction of stop codons within thenac open reading frame, were tested for the ability to activate hut and repress gdh in vivo. Most of the NACK and NACE fragments with 100 or more amino acids (wild-type NACK and NACE both have 305 amino acids) were functional in activating hut and repressing gdh expression in vivo. Full-length NACK and NACE were isolated as chimeric proteins with the maltose-binding protein (MBP). NACK and NACE released from such chimeras were able to activatehut transcription in a purified system in vitro, as were NACK129 and NACE100 (a NACKfragment of 129 amino acids and a NACE fragment of 100 amino acids) released from comparable chimeras. A set of NACE and NACK fragments carrying nickel-binding histidine tags (his6) at their C termini were also generated. All such constructs derived from NACE were insoluble, as was NACE itself. Of the his6-tagged constructs derived from NACK, NACK100 was inactive, but NACK120 was active. Several NAC fragments were tested for dimerization. NACK120-his6 and NACK100-his6 were dimers in solution. MBP-NACK and MBP-NACK129 were monomers in solution but dimerized when the MBP was released by cleavage with factor Xa. MBP-NACE was readily cleaved by factor Xa, but the resulting NACE was also degraded by the protease. However, MBP-NACE-his6 was completely resistant to cleavage by factor Xa, suggesting an interaction between the C and N termini of this protein.

scholarly journals TranSuite: a software suite for accurate translation and characterization of transcripts

2020 ◽  
Author(s):  
Juan C. Entizne ◽  
Wenbin Guo ◽  
Cristiane P.G. Calixto ◽  
Mark Spensley ◽  
Nikoleta Tzioutziou ◽  
...  
Keyword(s):  
Protein Translation ◽  
Software Suite ◽  
Transcript Isoforms ◽  
Nonsense Mediated Decay ◽  
Reading Frame ◽  
Premature Termination Codons ◽  
Translation Start ◽  
Protein Variants ◽  
Selection Of

ABSTRACTProtein translation programs often select the longest open reading frame (ORF) in a transcript leading to numerous inaccurate and mis-annotated ORFs in databases. Unproductive transcript isoforms containing premature termination codons (PTCs) are potential substrates for nonsense-mediated decay (NMD). These transcripts often contain truncated ORFs but are incorrectly annotated due to selection of a long ORF beginning at an AUG downstream of the PTC despite the transcript containing the authentic translation start AUG. In gene expression and alternative splicing analyses, it is important to identify transcript isoforms which code for different protein variants and to distinguish these from potential NMD substrates. Here, we present TranSuite, a pipeline of bioinformatics tools that address these challenges by performing accurate translations, characterizing alternative ORFs and identifying NMD and other features of transcripts in newly assembled and existing transcriptomes. Directly comparing ORFs defined by TranSuite and TransDecoder for the Arabidopsis transcriptome AtRTD2 identified ORF mis-calling in over 16k (27%) of transcripts by TransDecoder.

scholarly journals Engineering gene overlaps to sustain genetic constructs in vivo

2019 ◽  
Author(s):  
Antoine L. Decrulle ◽  
Antoine Frenoy ◽  
Thomas A. Meiller-Legrand ◽  
Aude Bernheim ◽  
Chantal Lotton ◽  
...  
Keyword(s):  
Fitness Landscape ◽  
Essential Gene ◽  
Loss Of Function ◽  
Evolutionary Potential ◽  
Reading Frame ◽  
E Coli ◽  
The Stability ◽  
Genetic Constructs ◽  
Selection Of

AbstractEvolution is often an obstacle to the engineering of stable biological systems due to the selection of mutations inactivating costly gene circuits. Gene overlaps induce important constraints on sequences and their evolution. We show that these constraints can be harnessed to increase the stability of synthetic circuits by purging loss-of-function mutations. We combine computational and synthetic biology approaches to rationally design an overlapping reading frame expressing an essential gene within an existing gene to protect. Our algorithm succeeded in creating overlapping reading frames in 80% of E. coli genes. Experimentally, scoring mutations in both genes of such overlapping construct, we found that a significant fraction of mutations impacting the gene to protect have a deleterious effect on the essential gene. Such an overlap thus protects a costly gene from removal by natural selection by associating the benefit of this removal with a larger or even lethal cost. In our synthetic constructs, the overlap converts many of the possible mutants into evolutionary dead-ends, effectively changing the fitness landscape and reducing the evolutionary potential of the system.

scholarly journals Escherichia coli mrsC Is an Allele ofhflB, Encoding a Membrane-Associated ATPase and Protease That Is Required for mRNA Decay

1998 ◽  
Vol 180 (7) ◽  
pp. 1929-1938 ◽  
Author(s):  
Rong-fu Wang ◽  
Eileen B. O’Hara ◽  
Marti Aldea ◽  
Cornelia I. Bargmann ◽  
Heather Gromley ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Atpase Activity ◽  
Mrna Decay ◽  
Biologically Active ◽  
Atp Binding ◽  
Binding Motif ◽  
Temperature Sensitive ◽  
Reading Frame ◽  
Amino Terminal

ABSTRACT The mrsC gene of Escherichia coli is required for mRNA turnover and cell growth, and strains containing the temperature-sensitive mrsC505 allele have longer half-lives than wild-type controls for total pulse-labeled and individual mRNAs (L. L. Granger et al., J. Bacteriol. 180:1920–1928, 1998). The cloned mrsC gene contains a long open reading frame beginning at an initiator UUG codon, confirmed by N-terminal amino acid sequencing, encoding a 70,996-Da protein with a consensus ATP-binding domain. mrsC is identical to the independently identifiedftsH gene except for three additional amino acids at the N terminus (T. Tomoyasu et al., J. Bacteriol. 175:1344–1351, 1993). The purified protein had a Km of 28 μM for ATP and a V max of 21.2 nmol/μg/min. An amino-terminal glutathione S-transferase–MrsC fusion protein retained ATPase activity but was not biologically active. A glutamic acid replacement of the highly conserved lysine within the ATP-binding motif (mrsC201) abolished the complementation of the mrsC505 mutation, confirming that the ATPase activity is required for MrsC function in vivo. In addition, themrsC505 allele conferred a temperature-sensitive HflB phenotype, while the hflB29 mutation promoted mRNA stability at both 30 and 44°C, suggesting that the inviability associated with the mrsC505 allele is not related to the defect in mRNA decay. The data presented provide the first direct evidence for the involvement of a membrane-bound protein in mRNA decay in E. coli.

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