Suppressor mutations define two regions in the Cbp1 protein important for mitochondrial cytochrome b mRNA stability in Saccharomyces cerevisiae

2003 ◽  
Vol 43 (5) ◽  
pp. 327-336 ◽  
Author(s):  
Maria A. Islas-Osuna ◽  
Timothy P. Ellis ◽  
Telsa M. Mittelmeier ◽  
Carol L. Dieckmann
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cytochrome B ◽  
Mrna Stability ◽  
Mitochondrial Cytochrome ◽  
Suppressor Mutations ◽  
Mitochondrial Cytochrome B ◽  
Cytochrome B Mrna

scholarly journals Expression, purification, crystallization and preliminary X-ray crystallographic studies of a mitochondrial membrane-associated protein Cbs2 from Saccharomyces cerevisiae

PeerJ ◽  
2021 ◽  
Vol 9 ◽  
pp. e10901
Author(s):  
Dan Wu ◽  
Guanyu Zhu ◽  
Yufei Zhang ◽  
Yan Wu ◽  
Chunlei Zhang ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Molecular Weight ◽  
Cytochrome B ◽  
Diffraction Data ◽  
Mitochondrial Cytochrome ◽  
Inner Membrane ◽  
X Ray ◽  
Mitochondrial Inner Membrane ◽  
Mitochondrial Cytochrome B ◽  
Cytochrome B Mrna

Background Mitochondria are unique organelles that are found in most eukaryotic cells. The main role of the mitochondria is to produce ATP. The nuclear genome encoded proteins Cbs1 and Cbs2 are located at the mitochondrial inner membrane and are reported to be essential for the translation of mitochondrial cytochrome b mRNA. Genetic studies show that Cbs2 protein recognizes the 5′ untranslated leader sequence of mitochondrial cytochrome b mRNA. However, due to a lack of biochemical and structural information, this biological process remains unclear. To investigate the structural characteristics of how Saccharomyces cerevisiae (S. cerevisiae) Cbs2 tethers cytochrome b mRNA to the mitochondrial inner membrane, a preliminary X-ray crystallographic study was carried out and is reported here. Methods The target gene from S. cerevisiae was amplified by polymerase chain reaction. The PCR fragment was digested by the NdeI and XhoI restriction endonucleases and then inserted into expression vector p28. After sequencing, the plasmid was transformed into Escherichia coli C43 competent cells. The selenomethionine derivative Cbs2 protein was overexpressed using M9 medium based on a methionine-biosynthesis inhibition method. The protein was first purified to Ni2+-nitrilotriacetate affinity chromatography and then further purified by Ion exchange chromatography and Gel-filtration chromatography. The purified Se-Cbs2 protein was concentrated to 10 mg/mL. The crystallization trials were performed using the sitting-drop vapor diffusion method at 16 °C. The complete diffraction data was processed and scaled with the HKL2000 package and programs in the CCP4 package, respectively. Results Cbs2 from S. cerevisiae was cloned, prokaryotic expressed and purified. The analysis of the size exclusion chromatography showed that the Cbs2 protein peaked at a molecular weight of approximately 90 KDa. The crystal belonged to the space group C2, with unit-cell parameters of a = 255.11, b = 58.10, c = 76.37, and β = 95.35°. X-ray diffraction data was collected at a resolution of 2.7 Å. The Matthews coefficient and the solvent content were estimated to be 3.22 Å 3 Da-1 and 61.82%, respectively. Conclusions In the present study Cbs2 from S. cerevisiae was cloned, expressed, purified, and crystallized for structural studies. The molecular weight determination results indicated that the biological assembly of Cbs2 may be a dimer.The preliminary X-ray crystallographic studies indicated the presence of two Cbs2 molecules in the asymmetric unit. This study will provide an experimental basis for exploring how Cbs2 protein mediates cytochrome b synthesis.

Ovarian mitochondrial cytochrome b mRNA levels increase with sexual maturity in freshwater eels (Anguilla spp.)

2003 ◽  
Vol 173 (1) ◽  
pp. 11-19 ◽  
Author(s):  
P. Lokman ◽  
Y. Kazeto ◽  
S. Ijiri ◽  
G. Young ◽  
T. Miura ◽  
...  
Keyword(s):  
Cytochrome B ◽  
Sexual Maturity ◽  
Mrna Levels ◽  
Mitochondrial Cytochrome ◽  
Mitochondrial Cytochrome B ◽  
Cytochrome B Mrna ◽  
Freshwater Eels

Editing of the grapevine mitochondrial cytochrome b mRNA and molecular modeling of the protein

Biochimie ◽  
2006 ◽  
Vol 88 (5) ◽  
pp. 431-435 ◽  
Author(s):  
María A. Islas-Osuna ◽  
Begonia Silva-Moreno ◽  
Nidia Caceres-Carrizosa ◽  
Jesús M. García-Robles ◽  
Rogerio R. Sotelo-Mundo ◽  
...  
Keyword(s):  
Molecular Modeling ◽  
Cytochrome B ◽  
Mitochondrial Cytochrome ◽  
Mitochondrial Cytochrome B ◽  
Cytochrome B Mrna

scholarly journals Genetic evidence for interaction between Cbp1 and specific nucleotides in the 5' untranslated region of mitochondrial cytochrome b mRNA in Saccharomyces cerevisiae.

1997 ◽  
Vol 17 (11) ◽  
pp. 6203-6211 ◽  
Author(s):  
W Chen ◽  
C L Dieckmann
Keyword(s):  
Cytochrome B ◽  
Mrna Stability ◽  
Untranslated Region ◽  
Base Change ◽  
Genetic Evidence ◽  
Site Directed Mutagenesis ◽  
Cis Element ◽  
Mitochondrial Cytochrome B ◽  
Single Base Change ◽  
Cytochrome B Mrna

The cytochrome b (COB) gene is encoded by the mitochondrial genome; however, its expression requires the participation of several nuclearly encoded protein factors. The yeast Cbp1 protein, which is encoded by the nuclear CBP1 gene, is required for the stabilization of COB mRNA. A previous deletion analysis identified an 11-nucleotide-long sequence within the 5' untranslated region of COB mRNA that is important for Cbp1-dependent COB mRNA stability. In the present study, site-directed mutagenesis experiments were carried out to define further the features of this cis element. The CCG sequence within this region was shown to be necessary for stability. A change in residue 533 of Cbp1 from aspartate to tyrosine suppresses the effects of a single-base change in the CCG element. This is strong genetic evidence that the nuclearly encoded Cbp1 protein recognizes and binds directly to the sequence containing CCG and thus protects COB mRNA from degradation.

Suppressor analyses of temperature-sensitive cbp1 strains of Saccharomyces cerevisiae: the product of the nuclear gene SOC1 affects mitochondrial cytochrome b mRNA post-transcriptionally.

Genetics ◽  
1994 ◽  
Vol 138 (3) ◽  
pp. 565-575
Author(s):  
R R Staples ◽  
C L Dieckmann
Keyword(s):  
Cytochrome B ◽  
Nuclear Gene ◽  
State Level ◽  
Temperature Sensitive ◽  
Restrictive Temperature ◽  
Gene Products ◽  
Mitochondrial Cytochrome ◽  
Mitochondrial Cytochrome B ◽  
Cytochrome B Mrna ◽  
Bypass Mechanism

Abstract The induction of mitochondrial function is dependent upon both nuclearly encoded and mitochondrially encoded gene products. To understand nuclear-mitochondrial interactions, we must first understand gene-specific interactions. The accumulation of mitochondrial cytochrome b (COB) RNA is dependent upon Cbp1p, encoded by the nuclear gene CBP1. Thus, respiration is dependent upon Cbp1p. In this study, suppressors of temperature-sensitive cbp1 (cbp1ts) strains were selected for restoration of respiratory capability at the restrictive temperature Ts+). One nuclearly encoded suppressor, extragenic to CBP1, is recessive with respect to the wild-type suppressor allele and is unlinked to other known genetic loci whose gene products are necessary for expression of COB mRNA. The suppressor, called soc1 for Suppressor of cbp1, suppresses several other cbp1ts alleles but does not operate via a bypass mechanism. Molecular analyses indicate that soc1 allows the steady-state level of COB mRNA to increase at high temperature but has little or no effect on the levels of COB pre-mRNA. These data have led us to propose that the product of the nuclear gene SOC1 is required for normal turnover of COB mRNA.

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