Resolution of the discrepancy between a gene translation - termination codon and the deduced sequence for release of the encoded polypeptide

The mature 109-amino-acid human platelet-derived growth factor B (PDGF-B) peptide is derived by intracellular processing from a 241-amino-acid precursor synthesized in mammalian cells, with removal of 81 N-terminal and 51 C-terminal amino acids. In order to produce directly the mature 109-amino acid PDGF-B peptide as a recombinant protein in Escherichia coli, a CGA codon at position 110 of a DNA sequence encoding the full-length precursor form of PDGF-B was converted into the translation termination codon TGA by in vitro mutagenesis. Expression of this DNA via a plasmid vector in E. coli resulted in production of two distinct PDGF-B proteins having apparent molecular masses of 15 and 19 kDa, with the latter species predominating. Structural characterization employing N- and C-terminal amino acid sequencing and MS analyses indicated that the 15 kDa protein is the expected 109-amino-acid PDGF-B, and that the 19 kDa protein represents a C-terminal extended PDGF-B containing 160 amino acids. Characterization of a unique tryptic peptide derived from the 19 kDa protein revealed that this longer form of PDGF-B results from mistranslation of the introduced TGA termination codon at position 110 as tryptophan, with translation subsequently proceeding to the naturally occurring TAG termination codon at position 161. Owing to the high rate of translation readthrough of TGA codons in this and occasionally other proteins, it appears that the use of TGA as a translation termination codon for proteins to be expressed in E. coli should be avoided when possible.

Download Full-text

Translation Termination Is Involved in Histone mRNA Degradation when DNA Replication Is Inhibited

Molecular and Cellular Biology ◽

10.1128/mcb.25.16.6879-6888.2005 ◽

2005 ◽

Vol 25 (16) ◽

pp. 6879-6888 ◽

Cited By ~ 43

Author(s):

Handan Kaygun ◽

William F. Marzluff

Keyword(s):

Dna Synthesis ◽

Translation Termination ◽

Termination Codon ◽

Histone Mrna ◽

Stem Loop ◽

Mrna Metabolism ◽

Histone Mrnas ◽

Iron Response Element ◽

Translation Termination Codon ◽

The Stability

ABSTRACT The levels of replication-dependent histone mRNAs are coordinately regulated with DNA synthesis. A major regulatory step in histone mRNA metabolism is regulation of the half-life of histone mRNAs. Replication-dependent histone mRNAs are the only metazoan mRNAs that are not polyadenylated. Instead, they end with a conserved stem-loop structure, which is recognized by the stem-loop binding protein (SLBP). SLBP is required for histone mRNA processing, as well as translation. We show here, using histone mRNAs whose translation can be regulated by the iron response element, that histone mRNAs need to be actively translated for their rapid degradation following the inhibition of DNA synthesis. We also demonstrate the requirement for translation using a mutant SLBP which is inactive in translation. Histone mRNAs are not rapidly degraded when DNA synthesis is inhibited or at the end of S phase in cells expressing this mutant SLBP. Replication-dependent histone mRNAs have very short 3′ untranslated regions, with the stem-loop located 30 to 70 nucleotides downstream of the translation termination codon. We show here that the stability of histone mRNAs can be modified by altering the position of the stem-loop, thereby changing the distance from the translation termination codon.

Download Full-text

Gene structure for the alpha 1 chain of a human short-chain collagen (type XIII) with alternatively spliced transcripts and translation termination codon at the 5' end of the last exon.

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.85.20.7491 ◽

1988 ◽

Vol 85 (20) ◽

pp. 7491-7495 ◽

Cited By ~ 38

Author(s):

L. Tikka ◽

T. Pihlajaniemi ◽

P. Henttu ◽

D. J. Prockop ◽

K. Tryggvason

Keyword(s):

Gene Structure ◽

Collagen Type ◽

Translation Termination ◽

Termination Codon ◽

Short Chain ◽

Alternatively Spliced ◽

Translation Termination Codon

Download Full-text

Nucleotide sequence of a macronuclear DNA molecule coding for α-tubulin from the ciliateStylonychia lemnae. Special codon usage: TAA is not a translation termination codon

Nucleic Acids Research ◽

10.1093/nar/13.2.415 ◽

1985 ◽

Vol 13 (2) ◽

pp. 415-433 ◽

Cited By ~ 114

Author(s):

E. Helftenbem

Keyword(s):

Nucleotide Sequence ◽

Codon Usage ◽

Translation Termination ◽

Termination Codon ◽

Dna Molecule ◽

Translation Termination Codon

Download Full-text

Structural Characterization of the Rous Sarcoma Virus RNA Stability Element

Journal of Virology ◽

10.1128/jvi.02113-08 ◽

2008 ◽

Vol 83 (5) ◽

pp. 2119-2129 ◽

Cited By ~ 23

Author(s):

Jason E. Weil ◽

Michalis Hadjithomas ◽

Karen L. Beemon

Keyword(s):

Secondary Structure ◽

Rous Sarcoma Virus ◽

Translation Termination ◽

Rna Stability ◽

Premature Termination Codon ◽

Termination Codon ◽

Stem Loop ◽

Future Design ◽

Rous Sarcoma ◽

Sarcoma Virus

ABSTRACT In eukaryotic cells, an mRNA bearing a premature termination codon (PTC) or an abnormally long 3′ untranslated region (UTR) is often degraded by the nonsense-mediated mRNA decay (NMD) pathway. Despite the presence of a 5- to 7-kb 3′ UTR, unspliced retroviral RNA escapes this degradation. We previously identified the Rous sarcoma virus (RSV) stability element (RSE), an RNA element downstream of the gag natural translation termination codon that prevents degradation of the unspliced viral RNA. Insertion of this element downstream of a PTC in the RSV gag gene also inhibits NMD. Using partial RNase digestion and selective 2′-hydroxyl acylation analyzed by primer extension (SHAPE) chemistry, we determined the secondary structure of this element. Incorporating RNase and SHAPE data into structural prediction programs definitively shows that the RSE contains an AU-rich stretch of about 30 single-stranded nucleotides near the 5′ end and two substantial stem-loop structures. The overall secondary structure of the RSE appears to be conserved among 20 different avian retroviruses. The structural aspects of this element will serve as a tool in the future design of cis mutants in addressing the mechanism of stabilization.

Download Full-text

A Premature Termination Codon Interferes with the Nuclear Function of an Exon Splicing Enhancer in an Open Reading Frame-Dependent Manner

Molecular and Cellular Biology ◽

10.1128/mcb.19.3.1640 ◽

1999 ◽

Vol 19 (3) ◽

pp. 1640-1650 ◽

Cited By ~ 17

Author(s):

Anand Gersappe ◽

David J. Pintel

Keyword(s):

Translation Termination ◽

Rna Stability ◽

Open Reading Frame ◽

Termination Codon ◽

Dependent Manner ◽

Enhancer Element ◽

Reading Frame ◽

Exon Splicing ◽

Nuclear Rna ◽

Splicing Enhancer

ABSTRACT Premature translation termination codon (PTC)-mediated effects on nuclear RNA processing have been shown to be associated with a number of human genetic diseases; however, how these PTCs mediate such effects in the nucleus is unclear. A PTC at nucleotide (nt) 2018 that lies adjacent to the 5′ element of a bipartite exon splicing enhancer within the NS2-specific exon of minute virus of mice P4 promoter-generated pre-mRNA caused a decrease in the accumulated levels of P4-generated R2 mRNA relative to P4-generated R1 mRNA, although the total accumulated levels of P4 product remained the same. This effect was seen in nuclear RNA and was independent of RNA stability. The 5′ and 3′ elements of the bipartite NS2-specific exon enhancer are redundant in function, and when the 2018 PTC was combined with a deletion of the 3′ enhancer element, the exon was skipped in the majority of the viral P4-generated product. Such exon skipping in response to a PTC, but not a missense mutation at nt 2018, could be suppressed by frame shift mutations in either exon of NS2 which reopened the NS2 open reading frame, as well as by improvement of the upstream intron 3′ splice site. These results suggest that a PTC can interfere with the function of an exon splicing enhancer in an open reading frame-dependent manner and that the PTC is recognized in the nucleus.

Download Full-text

A role for DIS3L2 over human nonsense-mediated mRNA decay targets

10.1101/722702 ◽

2019 ◽

Cited By ~ 1

Author(s):

Paulo J. da Costa ◽

Juliane Menezes ◽

Margarida Saramago ◽

Juan F. García-Moreno ◽

Hugo A. Santos ◽

...

Keyword(s):

Translation Termination ◽

Full Length ◽

Nonsense Mediated Decay ◽

Endonucleolytic Cleavage ◽

Nonsense Mediated Mrna Decay ◽

Translation Termination Codon ◽

Life Analysis ◽

Mrna Half Life ◽

Premature Translation Termination Codon

ABSTRACTThe nonsense-mediated decay (NMD) pathway selectively degrades mRNAs carrying a premature translation-termination codon but also regulates the abundance of a large number of physiological mRNAs that encode full-length proteins. In human cells, NMD-targeted mRNAs are degraded by endonucleolytic cleavage and exonucleolytic degradation from both 5’ and 3’ ends. This is done by a process not yet completely understood that recruits decapping and 5’-to-3’ exonuclease activities, as well as deadenylating and 3’-to-5’ exonuclease exosome activities. In yeast, DIS3/Rrp44 protein is the catalytic subunit of the exosome, but in humans, there are three known paralogues of this enzyme: DIS3, DIS3L1, and DIS3L2. DIS3L1 and DIS3L2 exoribonucleases localize in the same compartment where NMD occurs, but little is known about their role in this process. In order to unveil the role of DIS3L2 in NMD, here we show that some NMD-targets accumulate in DIS3L2-depleted cells. mRNA half-life analysis further supports that these NMD-targets are in fact DIS3L2 substrates. Besides, we observed that DIS3L2 acts over full-length transcripts, through a process that also involves UPF1. Moreover, DIS3L2-mediated decay is dependent on the activity of the terminal uridylyl transferases Zcchc6/11 (TUT7/4). Together, our findings establish a role for DIS3L2 and uridylation in NMD.

Download Full-text

Intranuclear defect in beta-globin mRNA accumulation due to a premature translation termination codon

Blood ◽

10.1182/blood.v64.1.13.13 ◽

1984 ◽

Vol 64 (1) ◽

pp. 13-22 ◽

Cited By ~ 47

Author(s):

K Takeshita ◽

BG Forget ◽

A Scarpa ◽

EJ Jr Benz

Keyword(s):

Translation Termination ◽

Beta Globin ◽

Mrna Synthesis ◽

Globin Mrna ◽

Mrna Accumulation ◽

S1 Nuclease ◽

Translation Termination Codon ◽

Premature Translation Termination ◽

Premature Translation Termination Codon

Abstract We have analyzed a cloned beta O-thalassemia (beta O-thal) gene from a patient doubly heterozygous for hemoglobin Lepore and beta O- thalassemia. Studies of 3H-uridine incorporation into beta-globin mRNA in this patient's erythroblasts suggested an intranuclear defect in both beta and Lepore (delta beta) mRNA synthesis, as did S1 nuclease analysis of nuclear RNA. However, the nucleotide sequence of the beta O- thal gene revealed only a single base change in codon 39 (CAG----UAG), which created a premature translation termination codon. The 5′ flanking sequence, including transcription promotor boxes and the mRNA initiation (CAP) site, were normal. The unexpected effect of this mutation on intranuclear beta-mRNA synthesis in vivo was studied by insertion of the cloned gene into a plasmid expression vector and transfection into tissue culture (COS-1) cells. beta-Globin mRNA produced by the transfected cells was assessed by S1 nuclease analysis. The beta O-39 thalassemia gene generated five- to tenfold less beta- mRNA than a normal beta-gene in both nuclear and cytoplasmic RNA, simulating the results observed in vivo. Moreover, the small amount of beta O-39 mRNA produced was as stable as normal beta-mRNA during an actinomycin D chase, ruling out rapid cytoplasmic turnover as a cause of the reduced accumulation. Cotransfection of the beta O-39 thalassemia gene with a mutant tyrosine suppressor tRNA gene resulted in restoration of the beta O-39 mRNA accumulation to near-normal levels. On the basis of these results, we suggest that the low levels of beta-mRNA known to exist in the common form of beta O-thalassemia, beta O-39 thalassemia, result from a lesion in transcription, or early posttranscriptional processes; the defect appears to be corrected by restoration of proper translational potential to the mutant mRNA, at least in a gene transfer-expression system in tissue-culture cells.

Download Full-text

Characterization of the complete mitochondrial genome of Metastrongylus salmi (M. salmi) derived from Tibetan pigs in Tibet, China

Acta Parasitologica ◽

10.1515/ap-2018-0032 ◽

2018 ◽

Vol 63 (2) ◽

pp. 280-286 ◽

Cited By ~ 1

Author(s):

Kun Li ◽

Muhammad Shahzad ◽

Hui Zhang ◽

Khalid Mehmood ◽

Xiong Jiang ◽

...

Keyword(s):

Dna Sequences ◽

Phylogenetic Trees ◽

Translation Termination ◽

Complete Mitochondrial Genome ◽

Nucleotide Composition ◽

Phylogenetic Position ◽

Protein Coding ◽

Tibetan Pigs ◽

Translation Termination Codon ◽

Rna Genes

AbstractThe present study was designed to determine and analyze themtgenomes ofMetastrongylus salmi(M.salmi), and reveal the phylogenetic relationships of this parasite usingmtDNA sequences. Results showed that the completemtgenome ofM.salmiwas 13722 bp containing 12 protein-coding genes (cox1-3, nad1-6, nad4L, atp6 and cytb), 22 transfer RNA genes, and 2 ribosomal RNA genes (rrnL and rrnS). The overall A+T content was 73.54% and the nucleotide composition was A (23.52%), C (6.14%), G (19.60%), T (50.02%), and N (UCAG) (0.73%). A total of 4237 amino acids are encoded from the Tibetan isolates ofM. salmi mtgenomes. The ATA was predicted as the most common starting codon with 41.7% (5/12 protein genes); and 11 of the 12 protein genes were found to have a TAG or TAA translation termination codon. By clustering together the phylogenetic trees of TibetanM.salmiand AustrianM.salmi, theM.salmiisolated from Tibetan pigs was found to be highly homological with that stemmed from Austrian one. This information provides meaningful insights into the phylogenetic position of theM.salmiChina isolate and represents a useful resource for selecting molecular markers for diagnosis and population studies.

Download Full-text