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Z Hillel  C W Wu 《Biochemistry》1977,16(15):3334-3342
The quaternary structures of Escherichia coli DNA-dependent RNA polymerase holenzyme (alpha 2 beta beta' sigma) and core enzyme (alpha 2 beta beta') have been investigated by chemical cross-linking with a cleavable bifunctional reagent, methyl 4-mercaptobutyrimidate, and noncleavable reagents, dimethyl suberimidate and N,N'-(1,4-phenylene)bismaleimide. A model of the subunit organization deduced from cross-linked subunit neighbors identified by dodecyl sulfate-polyacrylamide gel electrophoresis indicates that the large beta and beta' subunits constitute the backbone of both core and holoenzyme, while sigma and two alpha subunits interact with this structure along the contact domain of beta and beta' subunits. In holoenzyme, sigma subunit is in the vicinity of at least one alpha subunit. The two alpha subunits are close to each other in holoenzyme, core enzyme, and the isolated alpha 2 beta complex. Cross-linking of the "premature" core and holoenzyme intermediates in the in vitro reconstitution of active enzyme from isolated subunits suggests that these species are composed of subunit complexes of molecular weight lower than that of native core and holoenzyme, respectively. The structural information obtained for RNA polymerase and its subcomplexes has important implications for the enzyme-promoter recognition as well as the mechanism of subunit assembly of the enzyme.  相似文献   

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A series of seven carboxy-terminal deletion mutants of oat phytochrome A were stably expressed in transgenic tobacco to localize phytochrome domains involved in chromophore attachment, spectral integrity, photoreversibility between the red light (Pr)- and far-red light (Pfr)-absorbing forms, dimerization, and biological activity. Amino acids necessary for chromophore attachment in vivo were localized to the amino-terminal 398 residues because mutant proteins this small had covalently bound chromophore. Deletion mutants from the carboxy terminus to residue 653 were spectrally indistinguishable from the full-length chromoprotein. In contrast, further truncation to residue 399 resulted in a chromoprotein with a bleached Pfr absorbance spectrum, Pr and Pfr absorbance maxima shifted toward shorter wavelengths, and reduced Pfr to Pr phototransformation efficiency. Thus, residues between 399 ad 652 are required for spectral integrity but are not essential for chromophore attachment. The sequence(s) between residues 919 and 1093 appears to be necessary for dimerization. Carboxy-terminal mutants containing this region behaved as dimers under nondenaturing conditions in vitro, whereas truncations without this region behaved as monomers. None of the plants expressing high levels of deletion mutants lacking the 35 carboxy-terminal amino acids displayed the light-exaggerated phenotype characteristic of plants expressing biologically active phytochrome A, even when the truncated phytochromes were expressed at levels 6- to 15-fold greater than that effective for the full-length chromoprotein. Collectively, these data show that the phytochrome protein contains several separable carboxy-terminal domains required for structure/function and identify a domain within 35 residues of the carboxy terminus that is critical for the biological activity of the photoreceptor in vivo.  相似文献   

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FRS1 and FRS2, the structural genes encoding the large (alpha) and small (beta) subunits of yeast phenylalanyl-tRNA synthetase (PheRS) were placed under the control of the lacZ promoter by creating an artificial operon. The FRS2 gene was fused next to the promoter, followed by a 14 base pair intergenic sequence containing a translation reinitiation site in front of the FRS1 coding sequences. The engineered PheRS has 16 N-terminal amino acids from beta-galactosidase fused to the beta subunit. However, the purified protein shows a Km value for tRNA(Phe) that is indistinguishable from that of the the native enzyme. The product of the FRS2-FRS1 operon is not able to complement thermosensitive E. coli PheRS, indicating the lack of heterologous aminoacylation in vivo. We made a deletion in the FRS2 gene that removed about 150 amino terminal residues of the beta subunit. The truncated protein showed intact ATP-PPi exchange, whereas tRNA aminoacylation was lost. This result is similar to that of limited proteolysis performed on the native enzyme that yielded a tetrameric alpha 2 beta'2 structure, able to form aminoacyladenylate but unable to bind tRNA(Phe). A deletion of 50 amino acids from the carboxyl terminus of the beta chain resulted in the loss of both enzyme activities; this suggests the participation of the C-terminal end of the beta subunit in the active site or in subunit assembly to yield a tetrameric functional enzyme.  相似文献   

6.
The alpha subunit of Escherichia coli DNA-dependent RNA polymerase is encoded by the rpoA gene and plays a major role in enzyme assembly. A set of C-terminal deletion mutations of the rpoA gene was constructed. The results of mixed reconstitution experiments in vitro, using the truncated alpha polypeptides encoded by the rpoA deletion mutants, suggest that the amino-terminal two-thirds of alpha subunit is sufficient for the formation of pseudo-core complexes containing both beta and beta' subunits.  相似文献   

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We describe the cloning and sequence analysis of the region surrounding the gene for the alpha subunit of RNA polymerase from Chlamydia trachomatis. This region contains genes for proteins in the order SecY, S13, S11, alpha, and L17, which are equivalent to Escherichia coli and Bacillus subtilis r proteins. The incorporation of chlamydial alpha subunit protein into the E. coli RNA polymerase holoenzyme rather than its truncated variant lacking the amino terminus suggests the existence of structural conservation among alpha subunits from distantly related genera.  相似文献   

8.
Multisubunit RNA polymerase is an essential enzyme for regulated gene expression. Here we report two Escherichia coli RNA polymerase structures: an 11.0 A structure of the core RNA polymerase and a 9.5 A structure of the sigma(70) holoenzyme. Both structures were obtained by cryo-electron microscopy and angular reconstitution. Core RNA polymerase exists in an open conformation. Extensive conformational changes occur between the core and the holoenzyme forms of the RNA polymerase, which are largely associated with movements in ss'. All common RNA polymerase subunits (alpha(2), ss, ss') could be localized in both structures, thus suggesting the position of sigma(70) in the holoenzyme.  相似文献   

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Using a deletion approach on the alpha subunit of DNA polymerase III from Escherichia coli, we show that there is an N-proximal polymerase domain which is distinct from a more C-proximal tau and beta binding domain. Although deletion of 60 residues from the alpha N terminus abolishes polymerase activity, deletions of 48, 169, and 342 amino acids from the C terminus progressively impair its catalytic efficiency but preserve an active site. Deletion of 342 C-terminal residues reduces k(cat) 46-fold, increases the Km for gapped DNA 5.5-fold, and increases the Km for deoxynucleoside triphosphates (dNTPs) twofold. The 818-residue protein with polymerase activity displays typical Michaelis-Menten behavior, catalyzing a polymerase reaction that is saturable with substrate and linear with time. With the aid of newly acquired sequences of the polymerase III alpha subunit from a variety of organisms, candidates for two key aspartate residues in the active site are identified at amino acids 401 and 403 of the E. coli sequence by inspection of conserved acidic amino acids. The motif Pro-Asp-X-Asp, where X is a hydrophobic amino acid, is shown to be conserved among all known DnaE proteins, including those from Bacillaceae, cyanobacteria, Mycoplasma, and mycobacteria. The E. coli DnaE deletion protein with only the N-terminal 366 amino acids does not have polymerase activity, consistent with the proposed position of the active-site residues.  相似文献   

12.
The influenza virus hemagglutinin (HA) glycoprotein synthesized from cloned DNA in a simian virus 40 vector is expressed on the surface of infected primate cells. Previously, it has been demonstrated that mutant HAs lacking the hydrophobic carboxy terminus fail to anchor on the cell surface and therefore are secreted extracellularly. During analysis of additional HA deletion mutants derived from an HA-simian virus 40 recombinant, we found a mutant with an altered hydrophobic carboxy terminus that exhibited another phenotype. This deletion mutant, dl-12, produced HA that was neither secreted nor expressed on the infected cell surface. The mutant HA was similar to the wild-type HA in apparent molecular weight and extent of glycosylation as assayed by endoglycosidase H sensitivity. The mutant HA localized near the perinuclear region of infected cells as indicated by an indirect immunofluorescence assay. Sequence analysis showed that a 5-base-pair deletion had occurred before the region encoding the hydrophobic carboxy terminus. Nevertheless, the physicochemical properties of the wild-type HA carboxy terminus were maintained in that the truncated HA carboxy terminus consisted of predominantly hydrophobic amino acids followed by several charged amino acids residues. This similarity in the carboxy terminus between the wild-type and mutant HAs may be responsible for the functional similarities observed. In spite of these similarities, the mutant HA failed to mature at the surface. These results suggest that the maturation of the mutant HA is blocked during a late stage in the transit to the cell surface.  相似文献   

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In analogy to the Escherichia coli replicative DNA polymerase III we define two forms of DNA polymerase alpha: the core enzyme and the holoenzyme. The core enzyme is not able to elongate efficiently primed single-stranded DNA templates, in contrast to the holoenzyme which functions well on in vivo-like template. Using these criteria, we have identified and partially purified DNA polymerase alpha holoenzyme from calf thymus and have compared it to the corresponding homogeneous DNA polymerase alpha (defined as the core enzyme) from the same tissue. The holoenzyme is able to use single-stranded parvoviral DNA and M13 DNA with a single RNA primer as template. The core enzyme, on the other hand, although active on DNAs treated with deoxyribonuclease to create random gaps, is unable to act on these two long, single-stranded DNAs. E. coli DNA polymerase III holoenzyme also copies the two in vivo-like templates, while the core enzyme is virtually inactive. The homologous single-stranded DNA-binding proteins from calf thymus and from E. coli stimulate the respective holoenzymes and inhibit the core enzymes. These results suggest a cooperation between a DNA polymerase holoenzyme and its homologous single-stranded DNA-binding protein. The prokaryotic and the mammalian holoenzyme behave similarly in several chromatographic systems.  相似文献   

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A key step in the regulation of heat shock genes in Escherichia coli is the stress-dependent degradation of the heat shock promoter-specific sigma(32) subunit of RNA polymerase by the AAA protease, FtsH. Previous studies implicated the C termini of protein substrates, including sigma(32), as degradation signals for AAA proteases. We investigated the role of the C terminus of sigma(32) in FtsH-dependent degradation by analysis of C-terminally truncated sigma(32) mutant proteins. Deletion of the 5, 11, 15, and 21 C-terminal residues of sigma(32) did not affect degradation in vivo or in vitro. Furthermore, a peptide comprising the C-terminal 21 residues of sigma(32) was not degraded by FtsH in vitro and thus did not serve as a recognition sequence for the protease, while an unrelated peptide of similar length was efficiently degraded. The truncated sigma(32) mutant proteins remained capable of associating with DnaK and DnaJ in vitro but showed intermediate (5-amino-acid deletion) and strong (11-, 15-, and 21-amino-acid deletions) defects in association with RNA polymerase in vitro and biological activity in vivo. These results indicate an important role for the C terminus of sigma(32) in RNA polymerase binding but no essential role for FtsH-dependent degradation and association of chaperones.  相似文献   

15.
As a step toward understanding the assembly of the hepatitis B virus (HBV) nucleocapsid at a molecular level, we sought to define the primary sequence requirements for assembly of the HBV core protein. This protein can self assemble upon expression in Escherichia coli. Applying this system to a series of C-terminally truncated core protein variants, we mapped the C-terminal limit for assembly to the region between amino acid residues 139 and 144. The size of this domain agrees well with the minimum length of RNA virus capsid proteins that fold into an eight-stranded beta-barrel structure. The entire Arg-rich C-terminal domain of the HBV core protein is not necessary for assembly. However, the nucleic acid content of particles formed by assembly-competent core protein variants correlates with the presence or absence of this region, as does particle stability. The nucleic acid found in the particles is RNA, between about 100 to some 3,000 nucleotides in length. In particles formed by the full-length protein, the core protein mRNA appears to be enriched over other, cellular RNAs. These data indicate that protein-protein interactions provided by the core protein domain from the N terminus to the region around amino acid 144 are the major factor in HBV capsid assembly, which proceeds without the need for substantial amounts of nucleic acid. The presence of the basic C terminus, however, greatly enhances encapsidation of nucleic acid and appears to make an important contribution to capsid stability via protein-nucleic acid interactions. The observation of low but detectable levels of nucleic acid in particles formed by core protein variants lacking the Arg-rich C terminus suggests the presence of a second nucleic acid-binding motif in the first 144 amino acids of the core protein. Based on these findings, the potential importance of the C-terminal core protein region during assembly in vivo into authentic, replication-competent nucleocapsids is discussed.  相似文献   

16.
Minimum requirements for protease activation of flavin pyruvate oxidase.   总被引:1,自引:0,他引:1  
B L Bertagnolli  L P Hager 《Biochemistry》1991,30(33):8131-8137
Previous investigations have shown that the catalytic efficiency (kcat/KM) of pyruvate oxidase can be enhanced 450-fold by chymotryptic cleavage of a 23-residue peptide (alpha-peptide) from the carboxy terminus of the enzyme. The minimum requirement for proteolytic activation has been investigated by exposing pyruvate oxidase to a variety of carboxypeptidases, either singly or in combination. The extent of carboxypeptidase hydrolysis was followed by analyzing the release of amino acids and by mass spectral analysis of the truncated alpha-peptides which were derived from the carboxypeptidase-treated preparations. The results indicate that the removal of 7 carboxy-terminal residues does not activate the enzyme whereas the removal of 10 or 11 residues produces activated pyruvate oxidase. Activation of pyruvate oxidase by endoproteinase Glu-C confirms the carboxypeptidase results. Endoproteinase Glu-C specificity predicts hydrolytic cleavage of the peptide bond between Glu-561 and Val-562 with the removal of 11 residues from the carboxy terminus of the enzyme.  相似文献   

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To elucidate the structural basis for membrane attachment of the alpha subunit of the stimulatory G protein (Gs alpha), mutant Gs alpha cDNAs with deletions of amino acid residues in the amino and/or carboxy termini were transiently expressed in COS-7 cells. The particulate and soluble fractions prepared from these cells were analyzed by immunoblot using peptide specific antibodies to monitor distribution of the expressed proteins. Transfection of mutant forms of Gs alpha with either 26 amino terminal residues deleted (delta 3-28) or with 59 amino terminal residues deleted (delta 1-59) resulted in immunoreactive proteins which localized primarily to the particulate fraction. Similarly, mutants with 10 (delta 385-394), 32 (delta 353-384), or 42 (delta 353-394) amino acid residues deleted from the carboxy terminus also localized to the particulate fraction, as did a mutant form of Gs alpha lacking amino acid residues at both the amino and carboxy termini (delta 3-28)/(delta 353-384). Mutant and wild type forms of Gs alpha demonstrated a similar degree of tightness in their binding to membranes as demonstrated by treatment with 2.5 M NaCl or 6 M urea, but some mutant forms were relatively resistant compared with wild type Gs alpha to solubilization by 15 mM NaOH or 1% sodium cholate. We conclude that: (a) deletion of significant portions of the amino and/or carboxyl terminus of Gs alpha is still compatible with protein expression; (b) deletion of these regions is insufficient to cause cytosolic localization of the expressed protein. The basis of Gs alpha membrane targeting remains to be elucidated.  相似文献   

19.
DNA-dependent RNA polymerase from Micrococcus luteus can be isolated from cell extracts after removal of an excess of nucleic acids by fractionation with ammonium sulfate, followed by two consecutive gel filtrations through agarose and chromatography on cellulose phospate. Either homogeneous holoenzyme or a mixture of core and holoenzyme is obtained in this way, as is indicated by electrophoresis in polyacrylamide gels in the absence of detergent, where core enzyme migrates ahead of holoenzyme. Homogeneous core enzyme can be isolated from holoenzyme by chromatography on DEAE-cellulose. Core enzyme contains the subunits alpha, beta and beta' previously described [U.I. Lill et al., (1975) Eur. J. Biochem. 52, 411-420] in a molar ratio of 2:1:1. Holoenzyme contains an additional subunit sigma of 80 000 molecular weight (molar subunit composition alpha2 betabeta' sigma) and two relatively small polypeptides (molecular weight 14 000 and 25 000, respectively). Subunit sigma may be isolated from holoenzyme by chromatography on DEAE-cellulose at pH 6.9 in the presence of low concentrations of glycerol. The behaviour of holoenzyme during sedimentation in a glycerol gradient at low ionic strength indicates its occurrence as a dimer of the alpha2betabeta'sigma-protomer, whereas the monomeric form is preferred by core enzyme. Holoenzyme is much more active than core enzyme in RNA synthesis on bacteriophage T4DNA as template. The activity of the latter is stimulated by isolated sigma. M. luteus sigma as well as holoenzyme enhances also the activity of core enzyme fro- Escherichia coli. The formation of a hybrid between micrococcal sigma and E. coli core polymerase is also suggested by the influence of sigma on the oligomerisation of the enzyme from E. coli.  相似文献   

20.
Characterization of a functional domain of human calpastatin   总被引:3,自引:0,他引:3  
Expression plasmids were constructed from the cDNA of human calpastatin to examine the contribution to the inhibition of calpain of highly conserved sequences in each of four repetitive domains. A series of deletion derivatives of domain 1 proteins, truncated at either the amino or carboxy terminus, were produced in E. coli. Deletion from the amino terminus past the amino terminal conserved sequence decreased the inhibition. When the middle conserved sequence, the M-sequence, was further deleted, no inhibition was detected, but deletion from the carboxy terminus past the carboxy terminal conserved sequence did not decrease the inhibition until the M-sequence was reached. Nuclear magnetic resonance and circular dichroism spectra showed that domain 1 has an unfolded structure. Peptides that contained the M-sequence and some neighboring sequences were synthesized to measure the minimum size of the inhibitory peptide, which was the M-sequence with the next six residues on the amino terminal side.  相似文献   

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