. Saccharomyces cerevisiae TFIIIB is a complex of three subunits, TBP, the TFIIB-related factor BRF, and the more loosely associated polypeptide ؆. Although human homologs for two of the TFIIIB subunits, the TATA box-binding protein TBP and the TFIIB-related factor BRF, have been characterized, a human homolog of yeast B؆ has not been described. Moreover, human BRF, unlike yeast BRF, is not universally required for RNA polymerase III transcription. In particular, it is not involved in transcription from the small nuclear RNA (snRNA)-type, TATA-containing, RNA polymerase III promoters. Here, we characterize two novel activities, a human homolog of yeast B؆, which is required for transcription of both TATA-less and snRNA-type RNA polymerase III promoters, and a factor equally related to human BRF and TFIIB, designated BRFU, which is specifically required for transcription of snRNA-type RNA polymerase III promoters. Together, these results contribute to the definition of the basal RNA polymerase III transcription machinery and show that two types of TFIIIB activities, with specificities for different classes of RNA polymerase III promoters, have evolved in human cells.
We have developed a simple procedure to incorporate an EDTA-metal complex at a rationally selected site within a full-length protein. Our procedure has two steps: In step 1, we use site-directed mutagenesis to introduce a unique solvent-accessible cysteine residue at the site of interest. In step 2, we derivatize the resulting protein with S-(2-pyridylthio)cysteaminyl-EDTA-metal, a novel aromatic disulfide derivative of EDTA-metal. We have used this procedure to incorporate an EDTA-iron complex at amino acid 2 of the helix-turn-helix motif of each of two helix-turn-helix motif sequence-specific DNA binding proteins, catabolite gene activator protein (CAP) and Cro, and we have analyzed EDTA-iron-mediated DNA affinity cleavage by the resulting protein derivatives. The CAP derivative cleaves DNA at base pair 2 of the DNA half-site in the protein-DNA complex, and the Cro derivative cleaves DNA at base pairs -3 to 5 of the DNA half-site in the protein-DNA complex. We infer that amino acid 2 of the helix-turn-helix motif of CAP is close to base pair 2 of the DNA half-site in the CAP-DNA complex in solution and that amino acid 2 of the helix-turn-helix motif of Cro is close to base pairs -3 to 5 of the DNA half-site in the Cro-DNA complex in solution.(ABSTRACT TRUNCATED AT 250 WORDS)
In Class I CAP‐dependent promoters, the DNA site for CAP is located upstream of the DNA site for RNA polymerase. In Class II CAP‐dependent promoters, the DNA site for CAP overlaps the DNA site for RNA polymerase, replacing the ‐35 site. We have used an ‘oriented heterodimers’ approach to identify the functional subunit of CAP at two Class I promoters having different distances between the DNA sites for CAP and RNA polymerase [CC(‐61.5) and CC(‐72.5)] and at one Class II promoter [CC(‐41.5)]. Our results indicate that transcription activation at Class I promoters, irrespective of the distance between the DNA sites for CAP and RNA polymerase, requires the activating region of the promoter‐proximal subunit of CAP. In striking contrast, our results indicate that transcription activation at Class II promoters requires the activating region of the promoter‐distal subunit of CAP.
We have developed a straightforward biochemical method to determine the orientation of the DNA binding motif of a sequence-specific DNA binding protein relative to the DNA site in the protein-DNA complex. The method involves incorporation of a photoactivatable crosslinking agent at a single site within the DNA binding motif of the sequence-specific DNA binding protein, formation of the derivatized protein-DNA complex, UV-irradiation of the derivatized protein-DNA complex, and determination of the nucleotide(s) at which crosslinking occurs. We have applied the method to catabolite gene activator protein (CAP). We have constructed and analyzed two derivatives of CAP: one having a phenyl azide photoactivatable crosslinking agent at amino acid 2 of the helix-turn-helix motif of CAP, and one having a phenyl azide photoactivatable crosslinking agent at amino acid 10 of the helix-turn-helix motif of CAP. The results indicate that amino acid 2 of the helix-turn-helix motif is dose to the top-strand nucleotides of base pairs 3 and 4 of the DNA half site in the CAP-DNA complex, and that amino acid 10 of the helix-turn-helix motif is close to the bottom-strand nucleotide of base pair 10 of the DNA half site in the CAP-DNA complex. For many sequence-specific DNA binding proteins, protein-DNA interaction is mediated by a conserved DNA binding motif: e.g., the helix-turn-helix motif, the homeodomain motif, the C2C2 zinc finger motif, the C2H2 zinc finger motif, the bZip motif, or the bHLH (helix-loop-helix) motif (reviewed in refs. 1 and 2). We have developed a straightforward biochemical method to determine the orientation of the DNA binding motif of a sequence-specific DNA binding protein relative to the DNA site in the protein-DNA complex. The method is useful in cases in which the three-dimensional structure of the DNA binding motif is known, or can be predicted based on sequence homology, but the threedimensional structure of the protein-DNA complex is not known. In this paper, we report the method and a test case in which we have applied the method to catabolite gene activator protein [CAP; also referred to as cAMP receptor protein (CRP)].The method involves covalently attaching a photoactivatable crosslinking agent-one capable of reacting with DNA nucleotides upon UV irradiation-to the DNA binding motifof the sequence-specific DNA binding protein under investigation. The method has two parts: (i) One covalently attaches a photoactivatable crosslinking agent to the DNA binding motif ofthe sequence-specific DNA binding protein at a single amino acid (amino acid x). One then forms the protein-DNA complex, UV irradiates the protein-DNA complex, and determines the nucleotide(s) at which crosslinking occurs. The results of i identify a nucleotide(s) close to amino acid x in the protein-DNA complex. (ii) One covalently attaches a photoactivatable crosslinking agent to the DNA binding motif of the sequence-specific DNA binding protein, at a different single amino acid (amino acid y). One then forms the protein-DNA comp...
Escherichia coli catabolite gene activator protein (CAP) is a helix-turn-helix motif sequence-specific DNA binding protein [de Crombrugghe, B., Busby, S. & Buc, H. (1984) Science 224, 831-838;and Pabo, C. & Sauer, R. (1984) Annu. Rev. Biochem. 53,. In this work, CAP has been converted into a site-specific DNA cleavage agent by incorporation of the chelator 1,10-phenanthroline at amino acid 10 of One approach to address these issues is to construct synthetic or semisynthetic site-specific DNA cleavage agents that have DNA recognition sites longer than those of the known type II restriction endonucleases. A complex consisting of a chelator and an appropriate metal is capable of cleaving DNA in an essentially random, sequence-independent fashion; examples include the 1,10-phenanthrolineCu(I) complex (2) and the EDTA-Fe(II) complex (3, 4). It is possible to target this DNA-cleavage activity to specific DNA recognition sites in double-stranded DNA by covalently attaching the chelator to a sequence-specific DNA binding molecule (5-9).
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