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61.
A domain of the Klenow fragment of Escherichia coli DNA polymerase I has polymerase but no exonuclease activity 总被引:11,自引:0,他引:11
The Klenow fragment of DNA polymerase I from Escherichia coli has two enzymatic activities: DNA polymerase and 3'-5' exonuclease. The crystal structure showed that the fragment is folded into two distinct domains. The smaller domain has a binding site for deoxynucleoside monophosphate and a divalent metal ion that is thought to identify the 3'-5' exonuclease active site. The larger C-terminal domain contains a deep cleft that is believed to bind duplex DNA. Several lines of evidence suggested that the large domain also contains the polymerase active site. To test this hypothesis, we have cloned the DNA coding for the large domain into an expression system and purified the protein product. We find that the C-terminal domain has polymerase activity (albeit at a lower specific activity than the native Klenow fragment) but no measurable 3'-5' exonuclease activity. These data are consistent with the hypothesis that each of the three enzymatic activities of DNA polymerase I from E. coli resides on a separate protein structural domain. 相似文献
62.
Correction for non-uniform phosphate labeling of E. coli and phage RNAs synthesized in vivo 总被引:3,自引:0,他引:3
cells grown to phosphate starvation incorporate 32PO4 unequally into the α position of the four ribonucleotide triphosphates during a short period of labeling. A method for determining the relative specific activities of nucleotides in RNA molecules synthesized under these conditions and correcting sequence data is described. 相似文献
63.
An intact Box C sequence in the U3 snRNA is required for binding of fibrillarin, the protein common to the major family of nucleolar snRNPs. 总被引:30,自引:5,他引:25
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The mammalian U3 snRNP is one member of a recently described family of nucleolar snRNPs which also includes U8, U13, U14, X and Y. All of these snRNPs are immunoprecipitable by anti-fibrillarin autoantibodies, suggesting the existence of a common binding site for the 34 kDa fibrillarin (Fb) protein. Two short nucleotide sequences, called Boxes C and D, present in each of these RNAs are the most likely sites for fibrillarin binding. We have developed a HeLa in vitro assembly system for binding of fibrillarin to human U3 snRNA. Reconstitution of the input RNA is specific in our assay since four of the other nucleolar small RNAs (U8, U13, X and Y) which have Boxes C and D become immunoprecipitable by anti-fibrillarin whereas two RNAs which lack these sequences (5S and 5.8S) do not. Deletion analyses of the U3 snRNA demonstrate that the presence of Box C but not Box D is required for fibrillarin binding. Moreover, seven single or double site-specific mutations in the U3 Box C abolish binding. The role of the Box C-fibrillarin interaction in the biogenesis of the Fb snRNPs is discussed. 相似文献
64.
Three pseudogenes for human U13 snRNA belong to class III. 总被引:1,自引:0,他引:1
The nucleotide sequences of three pseudogenes for the small nucleolar RNA, U13, were determined from three human DNA clones. The sequences are reported 50 bp 5' and 3' to each gene. These pseudogenes belong to class III because they contain dispersed mismatches when compared to the previously determined U13 RNA sequence, an adenine-rich region at the 3' end, and short imperfect repeats flanking the 5' end of the coding sequence and the 3' end of the adenine-rich region. 相似文献
65.
Human acidic ribosomal phosphoproteins P0, P1, and P2: analysis of cDNA clones, in vitro synthesis, and assembly. 总被引:41,自引:7,他引:34
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cDNA clones encoding three antigenically related human ribosomal phosphoproteins (P-proteins) P0, P1, and P2 were isolated and sequenced. P1 and P2 are analogous to Escherichia coli ribosomal protein L7/L12, and P0 is likely to be an analog of L10. The three proteins have a nearly identical carboxy-terminal 17-amino-acid sequence (KEESEESD(D/E)DMGFGLFD-COOH) that is the basis of their immunological cross-reactivity. The identities of the P1 and P2 cDNAs were confirmed by the strong similarities of their encoded amino acid sequences to published primary structures of the homologous rat, brine shrimp, and Saccharomyces cerevisiae proteins. The P0 cDNA was initially identified by translation of hybrid-selected mRNA and immunoprecipitation of the products. To demonstrate that the coding sequences are full length, the P0, P1, and P2 cDNAs were transcribed in vitro by bacteriophage T7 RNA polymerase and the resulting mRNAs were translated in vitro. The synthetic P0, P1, and P2 proteins were serologically and electrophoretically identical to P-proteins extracted from HeLa cells. These synthetic P-proteins were incorporated into 60S but not 40S ribosomes and also assembled into a complex similar to that described for E. coli L7/L12 and L10. 相似文献
66.
Each of the conserved sequence elements flanking the cleavage site of mammalian histone pre-mRNAs has a distinct role in the 3''-end processing reaction. 总被引:19,自引:5,他引:14
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To study the substrate requirements for the histone 3'-end processing reaction of mammalian histone pre-mRNAs, we created a set of mutations in the sequences flanking the processing site of a mouse H3 gene. We found that deletion of the downstream purine-rich element hypothesized to interact with U7 small nuclear RNA abolishes in vitro 3'-end processing. Somewhat surprisingly, however, mutations in the hairpin loop element which destabilize or destroy the secondary structure reduce but do not abolish 3'-end processing. This is in apparent contrast to results obtained for the sea urchin system, where both sequence elements appear to be absolutely required for 3'-end formation. 相似文献
67.
A model for the non-specific binding of catabolite gene activator protein to DNA 总被引:3,自引:1,他引:2
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The binding of E. coli catabolite gene activator protein (CAP) to non-specific sequences of DNA has been modelled as an electrostatic interaction between four basic side chains of the CAP dimer and the charged phosphates of DNA. Calculation of the electrostatic contribution to the binding free energy at various separations of the two molecules shows that complex formation is favored when CAP and DNA are separated by as much as 12 A. Thus, the long range electrostatic interactions may provide the initial energy for complex formation and also the correct relative orientation of CAP and DNA. The non-specific complex does not involve the penetration of amino acid side chains into the major grooves of DNA and permits 'sliding' of the protein along DNA, which would enhance the rate of association of CAP with the specific site as has been proposed previously for lac repressor. We propose that, as it 'slides', CAP is moving in and out of the major grooves in order to sample the DNA sequence. Recognition of the specific DNA site is achieved by a complementarity in structure and hydrogen bonding between amino acids and the edges of base pairs exposed in the major grooves of DNA. 相似文献
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