Biomedical subjects

S A Darst

Publications and source records attributed to S A Darst.

At least 37 records · Page 2Linked to original sources

PubMed1997

A mutant RNA polymerase that forms unusual open promoter complexes.

We describe a mutant Escherichia coli RNA polymerase (RNAP) that forms stable open promoter complexes even at -20 degrees C but with a shortened melted region that extends downstream to only position -7. In the presence of initiating transcription substrates, the mutant RNAP undergoes a temperature-dependent isomerization, resulting in a promoter complex that is indistinguishable from the wild-type RNAP-promoter complex, with the melted region extended downstream to position +4. We propose that the open complex formed by the mutant RNAP represents an intermediate on the normal promoter-opening pathway and that our results support earlier findings that initial promoter opening occurs in the upstream region of the -10 promoter consensus element and subsequently extends downstream to encompass the transcription start site.

Base Sequence↗

PubMed1997

Tethering of the large subunits of Escherichia coli RNA polymerase.

The rpoB and rpoC genes of eubacteria and archaea, coding, respectively, for the beta and beta'-like subunits of DNA-dependent RNA polymerase, are organized in an operon with rpoB always preceding rpoC. Here, we show that in Escherichia coli the two genes can be fused and that the resulting 2751-amino acid beta::beta' fusion polypeptide assembles into functional RNA polymerase in vivo and in vitro. The results establish that the C terminus of the beta subunit and the N terminus of the beta' subunit are in close proximity to each other on the surface of the assembled RNA polymerase during all phases of the transcription cycle and also suggest that RNA polymerase assembly in vivo may occur co-translationally.

DNA-Directed RNA Polymerases↗

PubMed1997

Determinants for Escherichia coli RNA polymerase assembly within the beta subunit.

We used binding assays and other approaches to identify fragments of the Escherichia coli RNAP beta subunit involved in the obligatory interaction with the alpha subunit to form the stable assembly intermediate alpha2beta as well as in the interaction to recruit the beta' subunit into the alpha2beta sub-assembly. We show that two regions of evolutionarily conserved sequence near the C terminus of beta (conserved regions H and I) are central to the assembly of RNAP and likely make subunit-subunit contacts with both alpha and beta'.

Binding Sites↗

PubMed1997

Domain organization of Escherichia coli transcript cleavage factors GreA and GreB.

The GreA and GreB proteins of Escherichia coli induce cleavage of the nascent transcript in ternary elongation complexes of RNA polymerase. Gre factors are presumed to have two biologically important and evolutionarily conserved functions: the suppression of elongation arrest and the enhancement of transcription fidelity. A three-dimensional structure of GreB was generated by homology modeling on the basis of the known crystal structure of GreA. Both factors display similar overall architecture and surface charge distribution, with characteristic C-terminal globular and N-terminal coiled-coil domains. One major difference between the two factors is the "basic patch" on the surface of the coiled-coil domain, which is much larger in GreB than in GreA. In both proteins, a site near the basic patch cross-links to the 3' terminus of RNA in the ternary transcription complex. GreA/GreB hybrid molecules were constructed by genetic engineering in which the N-terminal domain of one protein was fused to the C-terminal domain of the other. In the hybrid molecules, both the coiled-coil and the globular domains contribute to specific binding of Gre factors to RNA polymerase, whereas the antiarrest activity and the GreA or GreB specificity of transcript cleavage is determined by the N-terminal domain. These results implicate the basic patch of the N-terminal coiled-coil domain as an important functional element responsible for the interactions with nascent transcript and determining the size of the RNA fragment to be excised during the course of the cleavage reaction.

Amino Acid Sequence↗

PubMed1996

Domain organization of the Escherichia coli RNA polymerase sigma 70 subunit.

We used limited trypsin digestion to determine the domain organization of the Escherichia coli RNA polymerase sigma 70 subunit. Trypsin-resistant fragments containing sigma 70 conserved region 2 (sigma 70(2)), and carboxy-terminal fragments containing conserved regions 3 and 4 (sigma 70(3-4)) were identified by a combination of amino acid sequencing and mass spectrometry. The domains were studied for partial biochemical functions of sigma 70.sigma 70(2) bound core RNA polymerase competitively with intact sigma 70. In contrast to sigma 70(2) alone, the RNA polymerase holoenzyme formed with sigma 70(2) specifically bound a single-stranded DNA oligomer with a sequence corresponding to the non-template strand of the -10 promoter element (the Pribnow box). Sigma 70(2) also forms crystals that are suitable for X-ray analysis. Sigma 70(3-4) bound the T4 AsiA protein with high affinity. The epitope for T4 AsiA on sigma 70 was further localized to within sigma 70[551-608], comprising sigma conserved region 4.2.

Amino Acid Sequence↗

PubMed1996

Structural modules of the large subunits of RNA polymerase. Introducing archaebacterial and chloroplast split sites in the beta and beta' subunits of Escherichia coli RNA polymerase.

The beta and beta' subunits of Escherichia coli DNA-dependent RNA polymerase are highly conserved throughout eubacterial and eukaryotic kingdoms. However, in some archaebacteria and chloroplasts, the corresponding sequences are "split" into smaller polypeptides that are encoded by separate genes. To test if such split sites can be accommodated into E. coli RNA polymerase, subunit fragments encoded by the segments of E. coli rpoB and rpoC genes corresponding to archaebacterial and chloroplast split subunits were individually overexpressed. The purified fragments, when mixed in vitro with complementing intact RNA polymerase subunits, yielded an active enzyme capable of catalyzing the phosphodiester bond formation. Thus, the large subunits of eubacteria and eukaryotes are composed of independent structural modules corresponding to the smaller subunits of archaebacteria and chloroplasts.

Amino Acid Sequence↗

PubMed1996

Crystal structure of a sigma 70 subunit fragment from E. coli RNA polymerase.

The 2.6 A crystal structure of a fragment of the sigma 70 promoter specificity subunit of E. coli RNA polymerase is described. Residues involved in core RNA polymerase binding lie on one face of the structure. On the opposite face, aligned along one helix, are exposed residues that interact with the -10 consensus promoter element (the Pribnow box), including four aromatic residues involved in promoter melting. The structure suggests one way in which DNA interactions may be inhibited in the absence of RNA polymerase and provides a framework for the interpretation of a large number of genetic and biochemical analyses.

Amino Acid Sequence↗

PubMed1996

The C-terminal domain revealed in the structure of RNA polymerase II.

The location of the CTD in the structure of RNA polymerase II has been determined by electron crystallography at 16 A resolution. Difference maps between wild-type enzyme and that lacking the CTD, or with an antibody fragment bound in place of the CTD, disclose the site of attachment of the CTD to the polymerase. Appropriate display of the polymerase structure reveals the CTD as an element projecting from this site of attachment into solution. A low relative density and large volume of this element identify the CTD as a conformationally mobile region.

Amino Acid Sequence↗

PubMed1995

The beta subunit Rif-cluster I is only angstroms away from the active center of Escherichia coli RNA polymerase.

Ribonucleotide analogs bound in the initiating site of Escherichia coli RNA polymerase-promoter complex were cross-linked to the beta subunit. Using limited proteolysis and chemical degradation, the cross-link was mapped to a segment of beta between amino acids Val516 and Arg540. This region (Rif-cluster I) is known to harbor many rifampicin-resistant (RifR) mutations. The results demonstrate that Rif-culster I is part of the "5'-face" of the active center and provide structural basis for the long-known effects of RifR mutations on transcription initiation, elongation, and termination.

Binding Sites↗

PubMed1995

Three-dimensional structure of E. coli core RNA polymerase: promoter binding and elongation conformations of the enzyme.

The structure of E. coli core RNA polymerase (RNAP) has been determined to approximately 23 A resolution by three-dimensional reconstruction from electron micrographs of flattened helical crystals. The structure reveals extensive conformational changes when compared with the previously determined E. coli RNAP holoenzyme structure, but resembles the yeast RNAPII structure. While each of these structures contains a thumb-like projection surrounding a channel 25 A in diameter, the E. coli RNAP holoenzyme thumb defines a deep but open groove on the molecule, whereas the thumb of E. coli core and yeast RNAPII form part of a ring that surrounds the channel. This may define promoter-binding and elongation conformations of RNAP, as E. coli holoenzyme recognizes promoter sites on double-stranded DNA, while both E. coli core and yeast RNAPII are elongating forms of the polymerase and are incapable of promoter recognition.

Crystallography↗

PubMed1995

Streptolydigin-resistant mutants in an evolutionarily conserved region of the beta' subunit of Escherichia coli RNA polymerase.

Mutations conferring streptolydigin resistance onto Escherichia coli RNA polymerase have been found exclusively in the beta subunit (Heisler, L. M., Suzuki, H., Landick, R., and Gross, C. A. (1993) J. Biol. Chem. 268, 25369-25375). We report here the isolation of a streptolydigin-resistant mutation in the E. coli rpoC gene, encoding the beta' subunit. The mutation is the Phe793-->Ser substitution, which occurred in an evolutionarily conserved segment of the beta' subunit. The homologous segment in the eukaryotic RNA polymerase II largest subunit harbors mutations conferring alpha-amanitin resistance. Both streptolydigin and alpha-amanitin are inhibitors of transcription elongation. Thus, the two antibiotics may inhibit transcription in their respective systems by a similar mechanism, despite their very different chemical nature.

Amino Acid Sequence↗

PubMed1995

Assembly of functional Escherichia coli RNA polymerase containing beta subunit fragments.

The Escherichia coli rpoB gene, which codes for the 1342-residue beta subunit of RNA polymerase (RNAP), contains two dispensable regions centered around codons 300 and 1000. To test whether these regions demarcate domains of the RNAP beta subunit, fragments encoded by segments of rpoB flanking the dispensable regions were individually overexpressed and purified. We show that these beta-subunit polypeptide fragments, when added with purified recombinant beta', sigma, and alpha subunits of RNAP, reconstitute a functional enzyme in vitro. These results demonstrate that the beta subunit is composed of at least three distinct domains and open another avenue for in vitro studies of RNAP assembly and structure.

Cloning, Molecular↗

PubMed1995

The mechanism of protein crystal growth from lipid layers.

Two-dimensional (2D) crystals of proteins on lipid monolayers can initiate the formation of large three-dimensional (3D) crystals suitable for X-ray diffraction studies. The role of the 2D crystals in this process has not been firmly established. While it is likely that the 2D crystals serve as nuclei for epitaxial crystal growth, other mechanisms, such as non-specific nucleation induced by the high local concentration of the protein at the surface of the lipid layer, cannot be excluded. Using streptavidin as a model system, we have now firmly established that 3D crystal growth from 2D crystals on lipid layers occurs by epitaxy. We show that 2D crystals of streptavidin (space group C222) on biotinated lipid layers nucleate the growth of a 3D crystal form (space group I4I22) that possesses a structural similarity with the 2D crystal, but have no effect on the growth of 3D crystal forms (I222 and P2(1)) that are unrelated to the 2D crystal. At lower pH, a new 3D crystal form (space group P1), unrelated to the previously described 2D crystals, grew from lipid layers. This discovery initially raised concern about the validity of the epitaxial mechanism, but these concerns were alleviated with the subsequent discovery of a structurally related 2D P1 crystal that grew in similar solution conditions. Some parameters affecting epitaxial growth of both the P1 and I4I22 crystals were investigated, revealing several noteworthy features of the epitaxial growth. (1) 2D crystals are very effective nucleating agents; for instance, the P1 2D crystals can direct the growth of P1 3D crystals even under conditions that favour the growth of other crystal forms. (2) The epitaxial 3D crystal grow very rapidly and at amazingly low protein concentrations; P1 3D crystals can be grown from solutions as low as 10 microM streptavidin. (3) There is no obligate requirement for the deposition of pre-formed 2D crystals; lipid layers alone are equally effective at promoting epitaxial crystal growth.

Bacterial Proteins↗

PubMed1995

Crystal structure of the GreA transcript cleavage factor from Escherichia coli.

Transcription elongation factors stimulate the activity of DNA-dependent RNA polymerases by increasing the overall elongation rate and the completion of RNA chains. One group of such factors, which includes Escherichia coli GreA, GreB and eukaryotic SII (TFIIS), acts by inducing hydrolytic cleavage of the transcript within the RNA polymerase, followed by release of the 3'-terminal fragment. Here we report the crystal structure of GreA at 2.2 A resolution. The structure contains an amino-terminal domain consisting of an antiparallel alpha-helical coiled-coil dimer which extends into solution, reminiscent of the coiled coil in seryl-tRNA synthetases. A site near the tip of the coiled-coil 'finger' plays a direct role in the transcript cleavage reaction by contacting the 3'-end of the transcript. The structure exhibits an unusual asymmetric charge distribution which indicates the manner in which GreA interacts with the RNA polymerase elongation complex.

Amino Acid Sequence↗

PubMed1995

Epitaxial growth of protein crystals from two-dimensional crystals on lipid layers.

Two-dimensional crystals of proteins formed on lipid layers effectively nucleate the epitaxial growth of three-dimensional protein crystals. Crystals suitable for X-ray crystallography can be grown in this way more rapidly, and using substantially lower concentrations of protein and precipitants, than when using conventional methods.

Crystallization↗

PubMed1994

Crystallization of GreA, a transcript cleavage factor from Escherichia coli.

GreA is a 17.6 kDa protein from Escherichia coli that induces cleavage of the nascent transcript in the elongating complex of RNA polymerase, followed by release of the 3'-terminal fragment. Crystals of GreA have been obtained from polyethylene glycol 4000, 2-propanol and sodium citrate, pH 5.6 and have been propagated by a novel seeding procedure. The crystals diffract beyond 2 A resolution and belong to the orthorhombic space group P2(1)2(1)2(1), with cell dimensions a = 101.7 A, b = 42.22 A, c = 40.05 A and with one molecule in the asymmetric unit.

Bacterial Proteins↗

PubMed1994

The sigma subunit conserved region 3 is part of "5'-face" of active center of Escherichia coli RNA polymerase.

Ribonucleotide analogs bound in the initiating site of Escherichia coli RNA polymerase holoenzyme in open promoter complexes were cross-linked to the beta and sigma 70 subunits. Using limited proteolysis and chemical degradation, the cross-link site in sigma 70 was mapped to a segment between amino acids Glu508 and Met561 containing the C-terminal part of conserved region 3. This result, when reconciled with genetic data on the interaction of sigma 70 conserved regions 2 and 4 with the -10 and -35 promoter regions, respectively, allows us to model the orientation of the sigma 70 subunit domains within the open promoter complex.

Amino Acid Sequence↗

PubMed1994

Epitaxial growth of protein crystals on lipid layers.

The growth of three-dimensional protein crystals is seeded by two-dimensional crystals formed on lipid layers. Such crystallization occurs faster and at lower precipitant and protein concentrations than conventional crystal growth. This approach may also allow the crystallization of proteins that resist attempts at crystal growth by other means.

Bacterial Proteins↗