ABSTRACT
We describe our efforts to crystallize binary MCM1/DNA and ternary MATalpha2/MCM1/DNA complexes, including the unsuccessful attempts to crystallize MCM1/DNA complexes and the successful design of DNA crystal packing that resulted in high-resolution crystals of the MATalpha2/MCM1/DNA complex. We detail general procedures useful for preparing protein/DNA cocrystals, including improved methods for producing and purifying DNA-binding proteins and DNA fragments, for purifying protein/DNA complexes, and for controlling pH conditions during crystallization. We also describe the rational design of DNA for protein/DNA cocrystallization attempts, based on our analysis of how straight and bent DNA with single base-pair overhangs can pack end-to-end in a crystal.
Subject(s)
DNA, Fungal/chemistry , DNA, Fungal/metabolism , DNA-Binding Proteins/chemistry , DNA-Binding Proteins/metabolism , Homeodomain Proteins/chemistry , Homeodomain Proteins/metabolism , Repressor Proteins/chemistry , Repressor Proteins/metabolism , Saccharomyces cerevisiae/chemistry , Transcription Factors/chemistry , Transcription Factors/metabolism , Base Pairing/genetics , Base Sequence , Binding Sites , Crystallization , Crystallography, X-Ray , DNA, Fungal/genetics , DNA, Fungal/isolation & purification , DNA, Recombinant/genetics , DNA-Binding Proteins/genetics , DNA-Binding Proteins/isolation & purification , Escherichia coli/genetics , Fungal Proteins/chemistry , Fungal Proteins/genetics , Fungal Proteins/isolation & purification , Fungal Proteins/metabolism , Genes, Fungal/genetics , Homeodomain Proteins/genetics , Homeodomain Proteins/isolation & purification , Hydrogen-Ion Concentration , Minichromosome Maintenance 1 Protein , Models, Molecular , Molecular Weight , Oligodeoxyribonucleotides/chemistry , Oligodeoxyribonucleotides/genetics , Oligodeoxyribonucleotides/isolation & purification , Oligodeoxyribonucleotides/metabolism , Operator Regions, Genetic/genetics , Peptide Fragments/chemistry , Peptide Fragments/genetics , Peptide Fragments/metabolism , Plasmids/genetics , Protein Structure, Tertiary , Receptors, Mating Factor , Receptors, Peptide/genetics , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/isolation & purification , Recombinant Proteins/metabolism , Repressor Proteins/genetics , Repressor Proteins/isolation & purification , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae Proteins , Sepharose/analogs & derivatives , Sepharose/metabolism , Transcription Factors/genetics , Transcription Factors/isolation & purificationABSTRACT
The X-ray crystal structure of the transcription factor IIA (TFIIA) in complex with the TATA-box-binding protein (TBP) and TATA-element DNA is presented at 2.5 A resolution. TFIIA is composed of a beta-barrel and a four-helix bundle motif that together have a boot-like appearance. The beta-barrel extends the TBP beta-sheet and bridges over the DNA major groove immediately upstream of the TATA box. The four-helix bundle contributes substantially to the surface of the complex available for interaction with additional transcription factors.
