Gir2 is an uncharacterized protein of
Saccharomyces cerevisiae, containing a RWD/GI domain. In this work, we report the biophysical characterization of Gir2. His-tagged Gir2, expressed and purified ...from
Escherichia coli, showed an abnormally slow migration on SDS–PAGE. The yeast expressed protein behaves similarly. Using mass spectrometry and peptide mass fingerprinting we demonstrated that the protein has the expected molecular mass (34
kDa). EDC modification of carboxylate groups reverted the anomalous migration on SDS–PAGE. Size exclusion chromatography showed that Gir2 has a Stokes radius larger than expected. Gir2 is thermostable and lacks extensive structure, as determined by CD analysis. Based on these findings, we suggest that Gir2 is a representative of the growing group of “natively unfolded” proteins.
Germline mutations in cardiac-specific transcription factor genes have been associated with congenital heart disease (CHD) and the homeodomain transcription factor NKX2-5 is an important member of ...this group. Indeed, more than 40 heterozygous NKX2-5 germline mutations have been observed in individuals with CHD, and these are spread along the coding region, with many shown to impact protein function. In pursuit of understanding causes of CHD, we analyzed n = 49 cardiac biopsies from 28 patients and identified by direct sequencing two nonsynonymous NKX2-5 alterations affecting alanine 119, namely c.356C>A (p.A119E) and c.355G>T, (p.A119S), in patients with AVSD and HLHS, respectively. In functional assays, a significant reduction in transcriptional activities could be determined for the NKX2-5 variants. Importantly, in one family the mother, besides p.A119E, carried a synonymous mutant allele in the homeodomain (c.543G>A, p.Q181), and a synonymous dbSNP (c.63A>G, p.E21) in the transactivation domain of the protein, that were transmitted to the CHD daughter. The presence of these variants in-cis with the p.A119E mutation led to a further reduction in transcriptional activities. Such difference in activity may be in part related to reduced protein expression for the double variant c.356C>A and c.543G>A. We propose changes in mRNA stability and folding, due to a silent mutation and a dbSNP in the NKX2-5 coding region to contribute to the functional defect. Although the clinical significance of the NKX2-5 haplotype identified in the CHD patients remains to be ascertained, we provide evidence of an interaction of a dbSNP, with synonymous and nonsynonymous mutations to negatively impact NKX2-5 transcriptional activity.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Based on characteristic amino acid sequences of kinases that phosphorylate the α subunit of eukaryotic translation initiation factor 2 (eIF2α kinases), degenerate oligonucleotide primers were ...constructed and used to polymerase chain reaction-amplify from genomic DNA of Neurospora crassa a sequence encoding part of a putative protein kinase. With this sequence an open reading frame was identified encoding a predicted polypeptide with juxtaposed eIF2α kinase and histidyl-tRNA synthetase-related domains. The 1646 amino acid sequence of this gene, called cpc-3, showed 35% positional identity over almost the entire sequence with GCN2 of yeast, which stimulates translation of the transcriptional activator of amino acid biosynthetic genes encoded by GCN4. Strains disrupted for cpc-3 were unable to induce increased transcription and derepression of amino acid biosynthetic enzymes in amino acid-deprived cells. The cpc-3 mutation did not affect the ability to up-regulate mRNA levels of cpc-1, encoding theGCN4 homologue and transcriptional activator of amino acid biosynthetic genes in N. crassa, but the mutation abolished the dramatic increase of CPC1 protein level in response to amino acid deprivation. These findings suggest that cpc-3 is the functional homologue of GCN2, being required for increased translation of cpc-1 mRNA in amino acid-starved cells.
Genetic screens in Saccharomyces cerevisiae have identified the roles of ribosome components, tRNAs and translation factors in translational fidelity. These screens rely on the suppression of altered ...start codons, nonsense codons or frameshift mutations in genes involved in amino acid or nucleotide metabolism. Many of these genes are regulated by the General Amino Acid Control (GAAC) pathway. Upon amino acid starvation, the kinase GCN2 induces the GAAC cascade via increased translation of the transcriptional activator GCN4 controlled by upstream open reading frames (uORFs). Overexpression of the GCN2 or GCN4 genes enhances the sensitivity of translation fidelity assays that utilize genes regulated by GCN4, such as the suppression of a +1 insertion by S.cerevisiae translation elongation factor 1A (eEF1A) mutants. Paromomycin and the prion PSI+, which reduce translational fidelity, do not increase GCN4 expression to induce the suppression phenotype and in fact reduce derepression. eEF1A mutations that reduce translation, however, reduce expression of GCN4 under non-starvation conditions. These eEF1A mutants also reduce HIS4 mRNA expression. Taken together, this system improves in vivo strategies for the analysis of translational fidelity and further provides new information on the interplay among translation fidelity, altered elongation and translational control via uORFs.
Based on characteristic amino acid sequences of kinases that phosphorylate the α subunit of eukaryotic translation initiation
factor 2 (eIF2α kinases), degenerate oligonucleotide primers were ...constructed and used to polymerase chain reaction-amplify
from genomic DNA of Neurospora crassa a sequence encoding part of a putative protein kinase. With this sequence an open reading frame was identified encoding a
predicted polypeptide with juxtaposed eIF2α kinase and histidyl-tRNA synthetase-related domains. The 1646 amino acid sequence
of this gene, called cpc-3 , showed 35% positional identity over almost the entire sequence with GCN2 of yeast, which stimulates translation of the transcriptional
activator of amino acid biosynthetic genes encoded by GCN4 . Strains disrupted for cpc-3 were unable to induce increased transcription and derepression of amino acid biosynthetic enzymes in amino acid-deprived
cells. The cpc-3 mutation did not affect the ability to up-regulate mRNA levels of cpc-1 , encoding the GCN4 homologue and transcriptional activator of amino acid biosynthetic genes in N. crassa , but the mutation abolished the dramatic increase of CPC1 protein level in response to amino acid deprivation. These findings
suggest that cpc-3 is the functional homologue of GCN2 , being required for increased translation of cpc-1 mRNA in amino acid-starved cells.
Phenotypic and molecular studies of the mutation U142 indicate that the cpc-2+ gene is required to activate general amino acid control under conditions of amino acid limitation in the vegetative ...growth phase, and for formation of protoperithecia in preparation for the sexual phase of the life cycle of Neurospora crassa. The cpc-2 gene was cloned by complementation of the cpc-2 mutation in a his-2ts bradytrophic background. Genomic and cDNA sequence analysis indicated a 1636 bp long open reading frame interrupted by four introns. The deduced 316 amino acid polypeptide reveals 70% positional identity over its full length with G-protein beta-subunit-related polypeptides found in humans, rat (RACK1), chicken, tobacco and Chlamydomonas. With the exception of RACK1 the function of these proteins is obscure. All are entirely made up of seven WD-repeats. Expression studies of cpc-2 revealed one abundant transcript in the wild type; in the mutant its level is drastically reduced. In mutant cells transformed with the complementing sequence, the transcript level, enzyme regulation and female fertility are restored. In the wild type the cpc-2 transcript is down-regulated under conditions of amino acid limitation. With cpc-2 a new element involved in general amino acid control has been identified, indicating a function for a WD-repeat protein that belongs to a class that is conserved throughout the evolution of eukaryotes.
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