Subject(s)
DNA/administration & dosage , Oocytes/metabolism , Particle Size , Transfection , Animals , Female , Microspheres , Xenopus laevis , beta-Galactosidase/geneticsABSTRACT
Zinc fingers are usually associated with proteins that interact with DNA. Yet in two oocyte-specific Xenopus proteins, TFIIA and p43, zinc fingers are used to bind 5S RNA. One of these, TFIIIA, also binds the 5S RNA gene. Both proteins have nine zinc fingers that are nearly identical with respect to size and spacing. We have determined the relative affinities of groups of zinc fingers from TFIIIA for both 5S RNA and the 5S RNA gene. We have also determined the relative affinities of groups of zinc fingers from p43 for 5S RNA. The primary protein regions for RNA and DNA interaction in TFIIIA are located at opposite ends of the molecule. All zinc fingers from TFIIIA participate in binding 5S RNA, but zinc fingers from the C terminus have the highest affinity. N-terminal zinc fingers are essential for binding the 5S RNA gene. In contrast, zinc fingers at the amino terminus of p43 are essential for binding 5S RNA.
Subject(s)
RNA, Ribosomal, 5S/metabolism , Transcription Factors/metabolism , Zinc Fingers/physiology , Amino Acid Sequence , Animals , DNA Mutational Analysis , Female , Molecular Sequence Data , Oocytes/metabolism , Sequence Homology, Nucleic Acid , Transcription Factor TFIIIA , Transcription Factors/genetics , Xenopus , Zinc Fingers/geneticsABSTRACT
A kanamycin-resistance (KmR) cassette was modified to allow its introduction as a blunt-ended DNA fragment into target plasmids. Subsequent excision of the KmR cassette with ApaI and religation of the plasmid leaves behind six base pairs (5'-GGGCCC) that result in the insertion of two amino acids in the target protein.
Subject(s)
Codon , Kanamycin Resistance/genetics , Mutagenesis, Insertional/methods , Base Sequence , DNA , DNA-Directed RNA Polymerases/genetics , Molecular Sequence Data , Plasmids , Restriction Mapping , T-Phages/genetics , Viral ProteinsABSTRACT
Bacteriophages T7 and T3 encode DNA-dependent RNA polymerases that are 82% homologous, yet exhibit a high degree of specificity for their own promoters. A region of the RNA polymerase gene (gene 1) that is responsible for this specificity has been localized using two approaches. First, the RNA polymerase genes of recombinant T7 x T3 phage that had been generated in other laboratories in studies of phage polymerase specificity were characterized by restriction enzyme mapping. This approach localized the region that determines promoter specificity to the 3' end of the polymerase gene, corresponding to the carboxyl end of the polymerase protein distal to amino acid 623. To define more closely the region of promoter specificity, a series of hybrid T7/T3 RNA polymerase genes was constructed by in vitro manipulation of the cloned genes. The specificity of the resulting hybrid RNA polymerases in vitro and in vivo indicates that an interval of the polymerase that spans amino acids 674 to 752 (the 674 to 752 interval) contains the primary determinant of promoter preference. Within this interval, the amino acid sequences of the T3 and T7 enzymes differ at only 11 out of 79 positions. It has been shown elsewhere that specific recognition of T3 and T7 promoters depends largely upon base-pairs in the region from -10 to -12. An analysis of the preference of the hybrid RNA polymerases for synthetic T7 promoter mutants indicates that the 674 to 752 interval is involved in identifying this region of the promoter, and suggests that another domain of the polymerase (which has not yet been identified) may be involved in identifying other positions where the two consensus promoter sequences differ (most notably at position -15).
Subject(s)
DNA-Directed RNA Polymerases/genetics , Escherichia coli/genetics , Promoter Regions, Genetic , T-Phages/genetics , Amino Acid Sequence , DNA-Directed RNA Polymerases/isolation & purification , DNA-Directed RNA Polymerases/metabolism , Escherichia coli/enzymology , Genes, Viral , Molecular Sequence Data , Plasmids , Restriction Mapping , Sequence Homology, Nucleic Acid , T-Phages/enzymologyABSTRACT
In somatic cells of Xenopus, transcription of the TFIIIA gene initiates greater than 200 bp upstream from the start site used in oocytes. The resultant mRNA encodes a protein, S-TFIIIA, that is 22 amino acids longer at its amino terminus than the abundant form of TFIIIA in oocytes (O-TFIIIA). S-TFIIIA binds the 5S RNA gene and 5S RNA, and both O- and S-TFIIIA promote the formation of stable transcription complexes on oocyte-type 5S RNA genes in an oocyte nuclear extract. We have not found any functional difference between the two forms of TFIIIA. Different transcription start sites suggest differential promoter usage--one in oocytes that permits high levels of gene activity and another that is used in somatic cells for low-level TFIIIA mRNA synthesis.
Subject(s)
Transcription Factors/genetics , Xenopus laevis/genetics , Amino Acid Sequence , Animals , Base Sequence , Molecular Sequence Data , Oocytes/metabolism , Protein Biosynthesis/genetics , RNA, Messenger/chemistry , RNA, Ribosomal, 5S/genetics , Transcription Factor TFIIIA , Transcription, GeneticABSTRACT
A 5S RNA binding protein (p43) in Xenopus is a major constituent of oocytes and comprises part of a 42S ribonucleoprotein storage particle. We have cloned and sequenced p43 cDNA from X. laevis and X. borealis as well as the cDNA for X. borealis TFIIIA. Like TFIIIA, p43 has nine zinc fingers, seven of which are exactly the same size as their counterparts in TFIIIA. Amino acid homology between the two proteins is restricted mainly to conserved residues characteristic of zinc fingers. In contrast to TFIIIA, which binds specifically to both 5S RNA and 5S RNA genes, p43 binds exclusively to 5S RNA.
