RESUMO
Adenosine cyclic 3',5'-phosphate receptor protein (CRP or CAP) is a regulatory protein involved in the transcription of several operons in Escherichia coli. cAMP-independent, nonspecific complexes of CRP and DNA were investigated by photochemical cross-linking of the protein to nonspecific DNA, whose thymines are substituted by 5-bromouracil (BrUra). The cross-linked protein was completely digested by trypsin, and the covalently bound peptides were sequenced. We identified two regions of the protein in close contact with DNA: one in the C-terminal part, overlapping the canonical helix-turn-helix motif, and the other one in the N-terminal part, which is usually not considered to belong to the DNA-interacting domain of CRP. This result lead us to propose models for nonspecific interaction, where the DNA is in contact with both the N- and C-terminal parts of the protein.
Assuntos
Proteínas de Transporte/metabolismo , Proteína Receptora de AMP Cíclico , AMP Cíclico/farmacologia , DNA Bacteriano/metabolismo , Escherichia coli/genética , Luz , Receptores de AMP Cíclico/metabolismo , Sequência de Aminoácidos , Sítios de Ligação , Bromouracila/metabolismo , Proteínas de Transporte/química , Reagentes de Ligações Cruzadas , Dados de Sequência Molecular , Fotoquímica , Estrutura Secundária de Proteína , Receptores de AMP Cíclico/química , Tripsina/metabolismoRESUMO
Protein MC1 is the major chromosomal protein in methanosarcinaceae. Using photochemical crosslinking on 5-bromouracil-substituted DNA, we identified the region of the protein that interacts with it. This region is located in the C-terminal part of the polypeptide chain, and the crosslinked amino-acids are in the region 74-86. Tryptophan 74 is one of the amino-acids crosslinked to DNA.
Assuntos
Proteínas Arqueais , Proteínas de Bactérias/metabolismo , Bromouracila , Reagentes de Ligações Cruzadas , DNA/metabolismo , Methanosarcina/química , Ribonucleoproteínas/metabolismo , Sítios de Ligação , FotoquímicaRESUMO
4',6-Diamidino-2-phenylindole, DAPI, forms fluorescent complexes with DNA. This property has been used to quantify DNA on the basis of fluorometric test. However, the fluorescence quantum yield of DAPI increases also with tRNA. DNA estimation needs particular care in the presence of tRNA. For DNA containing 50% adenine-thymine (AT), DAPI can be used if DNA represents at least 3.4% of the total nucleotide material. This percentage varies with the AT/guanosine-cytosine content. When the fraction of DNA decreases further, the DAPI assay can no longer be used.
