Mo } ecular C } oning of groESL Locus , of Chaperonins , GroEL and
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چکیده
The groESL locus of a protein-hypersecreting bacterium, BaciULts brevis, was cloned by PCR using primers designed based on the DNA sequence of a B. subtilis homolog. GroEL protein was purified to apparent homogeneity and its ATPase activity was characterized; it hydrolyzed ATP, CTP, and TTP in this order of reaction rate, and its specific activity for ATP was O.lpmole!minfmg protein. Purified GroEL forms a tetradecamer. GroEL was estimated to contain 22% orhelix, 24% fi-sheet, and 19% turn structures, by CD measurement. GroES protein was also highly purified to examine its chaperonin activity. GroEL protected from thermal inactivation of and showed refolding-
منابع مشابه
A second groEL-like gene, organized in a groESL operon is present in the genome of Synechocystis sp. PCC 6803.
Using a groEL gene of Synechococcus sp. PCC 7942 as a DNA probe, a 4.8-kilobase pair (kbp) BamHI fragment of chromosomal DNA of Synechocystis sp. PCC 6803 was cloned. Sequencing of 3.25 kbp of the BamHI fragment revealed three open reading frames. The amino acid sequences deduced from the nucleotide sequences of the two open reading frames are identical to those gained from N-terminal sequencin...
متن کاملMycobacterium tuberculosis GroEL homologues unusually exist as lower oligomers and retain the ability to suppress aggregation of substrate proteins.
Chaperonin-60s are large double ring oligomeric proteins with a central cavity where unfolded polypeptides undergo productive folding. In conjunction with their co-chaperonin, Chaperonin-60s bind non-native polypeptides and facilitate their refolding in an ATP-dependent manner. The ATPase activity of Chaperonin-60 is tightly regulated by the 10 kDa co-chaperonin. In contrast to most other bacte...
متن کاملThe groESL operon of Agrobacterium tumefaciens: evidence for heat shock-dependent mRNA cleavage.
The heat shock response of the groESL operon of Agrobacterium tumefaciens was studied at the RNA level. The operon was found to be activated under heat shock conditions and transcribed as a polycistronic mRNA that contains the groES and groEL genes. After activation, the polycistronic mRNA appeared to be cleaved between the groES and groEL genes and formed two monocistronic mRNAs. The groES cle...
متن کاملCloning and characterization of the groESL operon from Bacillus subtilis.
The sequence of the 10 N-terminal amino acids of a Bacillus subtilis protein that cross-reacts with antibody to Escherichia coli GroEL was used to design a set of degenerate oligonucleotide probes. These probes identified a clone which carries almost the entire groESL operon from a B. subtilis subgenomic library. By chromosomal walking, an additional fragment carrying the 3' end of groESL and i...
متن کاملCo-overexpression of bacterial GroESL chaperonins partly overcomes non-productive folding and tetramer assembly of E. coli-expressed human medium-chain acyl-CoA dehydrogenase (MCAD) carrying the prevalent disease-causing K304E mutation.
The influence of co-overexpression of the bacterial chaperonins GroEL and GroES on solubility, tetramer formation and enzyme activity of three variants of heterologously-expressed human medium-chain acyl-CoA dehydrogenase (MCAD) was analysed in order to investigate the molecular mechanism underlying MCAD deficiency caused by the prevalent K304E mutation. Depending on which of the three amino ac...
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