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R Landick

Publications and source records attributed to R Landick.

At least 19 recordsLinked to original sources
PubMed1997

Preferential interaction of the his pause RNA hairpin with RNA polymerase beta subunit residues 904-950 correlates with strong transcriptional pausing.

RNA secondary structures (hairpins) that form as the nascent RNA emerges from RNA polymerase are important components of many signals that regulate transcription, including some pause sites, all rho-independent terminators, and some antiterminators. At the his leader pause site, a 5-bp-stem, 8-nt-loop pause RNA hairpin forms 11 nt from the RNA 3' end and stabilizes a transcription complex conformation slow to react with NTP substrate. This stabilization appears to depend at least in part on an interaction with RNA polymerase. We tested for RNA hairpin interaction with the paused polymerase by crosslinking 5-iodoUMP positioned specifically in the hairpin loop. In the paused conformation, strong and unusual crosslinking of the pause hairpin to beta904-950 replaced crosslinking to beta' and to other parts of beta that occurred in nonpaused complexes prior to hairpin formation. These changes in nascent RNA interactions may inhibit reactive alignment of the RNA 3' end in the paused complex and be related to events at rho-independent terminators.

Amino Acid Sequence↗
PubMed1997

Effects of neutral salts on RNA chain elongation and pausing by Escherichia coli RNA polymerase.

We examined the effects of neutral salts and the non-ionic solute 2-methyl,-2,4-pentanediol (MPD) on transcript elongation by Escherichia coli RNA polymerase and on pausing induced by the multipartite his leader pause signal. All solutes tested slowed the overall rate of elongation, with anions showing the dominant effects in the order: (most inhibitory) HPO4(2-) > OAc- > SO4(2-) > ClO4- > I- approximately NO3- > Br approximately Cl- approximately MPD (least inhibitory). Although the protein structure-stabilizing anions HPO4(2-), OAc-, and SO4(2-) also increased the pause half-life at the his leader pause site, the remaining solutes accelerated escape from pause site in the order: (greatest acceleration) NO3- > ClO4- > I- > Br- > Cl- > MPD (least acceleration). Cl(-)-induced acceleration of escape from the pause site also occurred on mutant templates altered for the 3'-proximal region, RNA 3' end, or downstream DNA. The effect was eliminated, however, by base substitutions that destabilize the pause RNA hairpin or that extend it toward the 3' end. This "perfect hairpin" itself reduced the pause half-life by a factor of 3. We suggest that the pause RNA hairpin stabilizes a paused conformation of the transcription complex through an interaction with an easily disordered region of RNA polymerase. Extending the stem of the pause hairpin may disrupt the interaction by altering the position of the hairpin in the transcription complex. Anions may either compete for the interaction directly or disorder the site of hairpin interaction by chaotropic effects. We suggest that the negative effect of structure-stabilizing anions like OAc- and SO4(2-) may reflect passage of RNA polymerase through significantly different conformations during rapid elongation, some of which may expose hydrophobic surface.

Anions↗
PubMed1997

Multiple interactions stabilize a single paused transcription intermediate in which hairpin to 3' end spacing distinguishes pause and termination pathways.

Transcription is delayed in the leader regions of the Escherichia coli trp and his operons by multipartite pause signals that consist of four components: a nascent RNA structure (the pause hairpin), the 10 or 11 nt 3'-proximal region between the pause hairpin and the RNA 3' end, the bases in the active site, and approximately 14 bp of duplex DNA downstream from the pause site. Results described in the accompanying paper suggest that the his pause hairpin slows nucleotide addition via interaction with an easily disordered surface on RNA polymerase. Here we report that the four pause signal components slow nucleotide addition in a single kinetic intermediate. Formation of the paused transcription complex, in contrast, involves synergistic effects of RNA and DNA sequences that select the wild-type pause site from among several adjacent possibilities. Extending the pause hairpin with one G x C base-pair reduces pausing, apparently by interfering with pause hairpin interaction; adding a second C x G base-pair that reduces the 3'-proximal RNA to 9 nt or less (within the 7 to 9 nt characteristic of rho-independent terminators) induces transcript release. We propose that escape from the pause is governed by a rate-limiting isomerization that may require substrate NTP binding to re-establish the active site geometry, whereas transcript release and termination ensue when the hairpin interaction is weakened and isomerization to an active conformation is blocked.

Bacterial Proteins↗
PubMed1997

Nuclease cleavage of the upstream half of the nontemplate strand DNA in an Escherichia coli transcription elongation complex causes upstream translocation and transcriptional arrest.

We tested the susceptibility of nucleic acid strands in a halted transcription elongation complex to digestion by micrococcal nuclease (MN). The 16-nucleotide nascent RNA was protected within RNA polymerase. A 27-28-nucleotide template strand DNA fragment also was resistant to MN digestion. However, the upstream half of the nontemplate DNA within this region was digested rapidly by MN, suggesting that the nontemplate strand emerges from the RNA polymerase near the middle of the melted transcription bubble with the bases oriented away from the enzyme surface. MN cleavage of the exposed nontemplate DNA shifted polymerase backward, making it unable to extend the RNA chain. However, the MN-trimmed G16 complexes could be reactivated by GreB-stimulated cleavage of the nascent RNA. These results favor a model of transcriptional arrest involving upstream slippage of RNA polymerase along the RNA and DNA chains. They also suggest that the exposed segment of nontemplate DNA may directly or indirectly stabilize the lateral position of the transcription complex along the DNA.

Base Sequence↗
PubMed1997

Stretching DNA with optical tweezers.

Force-extension (F-x) relationships were measured for single molecules of DNA under a variety of buffer conditions, using an optical trapping interferometer modified to incorporate feedback control. One end of a single DNA molecule was fixed to a coverglass surface by means of a stalled RNA polymerase complex. The other end was linked to a microscopic bead, which was captured and held in an optical trap. The DNA was subsequently stretched by moving the coverglass with respect to the trap using a piezo-driven stage, while the position of the bead was recorded at nanometer-scale resolution. An electronic feedback circuit was activated to prevent bead movement beyond a preset clamping point by modulating the light intensity, altering the trap stiffness dynamically. This arrangement permits rapid determination of the F-x relationship for individual DNA molecules as short as -1 micron with unprecedented accuracy, subjected to both low (approximately 0.1 pN) and high (approximately 50 pN) loads: complete data sets are acquired in under a minute. Experimental F-x relationships were fit over much of their range by entropic elasticity theories based on worm-like chain models. Fits yielded a persistence length, Lp, of approximately 47 nm in a buffer containing 10 mM Na1. Multivalent cations, such as Mg2+ or spermidine 3+, reduced Lp to approximately 40 nm. Although multivalent ions shield most of the negative charges on the DNA backbone, they did not further reduce Lp significantly, suggesting that the intrinsic persistence length remains close to 40 nm. An elasticity theory incorporating both enthalpic and entropic contributions to stiffness fit the experimental results extremely well throughout the full range of extensions and returned an elastic modulus of approximately 1100 pN.

DNA↗
PubMed1996

Amino acid substitutions in the two largest subunits of Escherichia coli RNA polymerase that suppress a defective Rho termination factor affect different parts of the transcription complex.

Among the earliest rpoBC mutations identified are three suppressors of the conditional lethal rho allele, rho201. These three mutations are of particular interest because, unlike rpoB8, they do not increase termination at all rho-dependent and rho-independent terminators. rpoB211 and rpoB212 both change Asn-1072 to His in conserved region H of rpoB (betaN1072H), whereas rpoC214 changes Arg-352 to Cys in conserved region C of rpoC (beta'R352C). Both substitutions significantly reduce the overall rate of transcript elongation in vitro relative to wild-type RNA polymerase; however, they probably slow elongation for different reasons. The nucleotide triphosphate concentrations required at the T7 A1 promoter for both abortive trinucleotide synthesis and for promoter escape are much greater for betaN1072H. In contrast, beta'R352C and two adjacent substitutions (beta'G351S and beta'S350F), but not betaN1072H, formed open complexes of greatly reduced stability. The sequence in this region of beta' modestly resembles a region of Escherichia coli DNA polymerase I that contacts the phosphate backbone of DNA in co-crystals. Core determinants affecting open complex formation do not reside exclusively in beta', however, since the Rifr mutation rpoB2 in beta also dramatically destabilized open complexes. We suggest that the principal defects of the two Rho-suppressing substitutions may differ, perhaps reflecting a greater role of beta region H in nucleoside triphosphate-binding and nucleotide addition and of beta' region C in contacts to the DNA strands that could be important for translocation. Although both probably suppress rho201 by slowing RNA chain elongation, these differences may lead to terminator specificity that depends on the rate-limiting step at different sites.

Amino Acid Sequence↗
PubMed1996

Isolation, purification, and in vitro characterization of recessive-lethal-mutant RNA polymerases from Escherichia coli.

The beta subunit of prokaryotic RNA polymerase shares significant sequence similarity with its eukaryotic and archaeal counterparts across most of the protein. Nine segments of particularly high similarity have been identified and are termed segments A through I. We have isolated severely defective Escherichia coli RNA polymerase mutants, most of which are unable to support bacterial growth. The majority of the substitutions affect residues in one of the conserved segments of beta, including invariant residues in segments D (amino acids 548 to 577), E (amino acids 660 to 678), and I (amino acids 1198 to 1296). In addition, recessive-lethal mutations that affect residues highly conserved only among prokaryotes were identified. They include a substitution in the extreme amino terminus of beta, a region in which no substitutions have previously been identified, and one rpoB mutation that truncates the polypeptide without abolishing minimal polymerase function in vitro. To examine the recessive-lethal alleles in vitro, we devised a novel method to remove nonmutant enzyme from RNA polymerase preparations by affinity tagging the chromosomal rpoB gene. In vitro examination of a subset of purified recessive-lethal RNA polymerases revealed that several substitutions, including all of those altering conserved residues in segment I, severely decrease transcript elongation and increase termination. We discuss the insights these mutants lend to a structure-function analysis of RNA polymerase.

Amino Acid Sequence↗
PubMed1995

Transcription against an applied force.

The force produced by a single molecule of Escherichia coli RNA polymerase during transcription was measured optically. Polymerase immobilized on a surface was used to transcribe a DNA template attached to a polystyrene bead 0.5 micrometer in diameter. The bead position was measured by interferometry while a force opposing translocation of the polymerase along the DNA was applied with an optical trap. At saturating nucleoside triphosphate concentrations, polymerase molecules stalled reversibly at a mean applied force estimated to be 14 piconewtons. This force is substantially larger than those measured for the cytoskeletal motors kinesin and myosin and exceeds mechanical loads that are estimated to oppose transcriptional elongation in vivo. The data are consistent with efficient conversion of the free energy liberated by RNA synthesis into mechanical work.

Biophysical Phenomena↗
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

Discontinuous movements of DNA and RNA in RNA polymerase accompany formation of a paused transcription complex.

A central enigma of transcriptional regulation is how the normally efficient transcription elongation complex stops at pause and termination signals. One possibility, raised by the discovery that RNA polymerase sometimes contracts its DNA footprint, is that discontinuous movements contribute to recognizing these signals. We report that E. coli RNA polymerase responds to sequences immediately downstream and upstream from the his leader pause site by changing neither its downstream DNA contact nor its upstream RNA contact for 8 bp preceding the pause. This compressed complex isomerizes to a paused conformation by an approximately 10 bp jump of its downstream DNA contact and simultaneous extrusion of an RNA hairpin that stabilizes the paused conformation. We suggest pausing and termination could be alternative outcomes of a similar isomerization that depend on the strength of contacts to 3'-proximal RNA remaining after the jump.

Base Sequence↗
PubMed1994

Termination-altering amino acid substitutions in the beta' subunit of Escherichia coli RNA polymerase identify regions involved in RNA chain elongation.

To identify regions of the largest subunit of RNA polymerase that are potentially involved in transcript elongation and termination, we have characterized amino acid substitutions in the beta' subunit of Escherichia coli RNA polymerase that alter expression of reporter genes preceded by terminators in vivo. Termination-altering substitutions occurred in discrete segments of beta', designated 2, 3a, 3b, 4a, 4b, 4c, and 5, many of which are highly conserved in eukaryotic homologs of beta'. Region 2 substitutions (residues 311-386) are tightly clustered around a short sequence that is similar to a portion of the DNA-binding cleft in E. coli DNA polymerase I. Region 3b (residues 718-798) corresponds to the segment of the largest subunit of RNA polymerase II in which amanitin-resistance substitutions occur. Region 4a substitutions (residues 933-936) occur in a segment thought to contact the transcript 3' end. Region 5 substitutions (residues 1308-1356) are tightly clustered in conserved region H near the carboxyl terminus of beta'. A representative set of mutant RNA polymerases were purified and revealed unexpected variation in percent termination at six different rho-independent terminators. Based on the location and properties of these substitutions, we suggest a hypothesis for the relationship of subunits in the transcription complex.

Amino Acid Sequence↗
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

GreA-induced transcript cleavage in transcription complexes containing Escherichia coli RNA polymerase is controlled by multiple factors, including nascent transcript location and structure.

The Escherichia coli GreA and GreB proteins induce cleavage of 3' fragments from nascent transcripts in halted transcription complexes. We have overproduced and purified the GreA protein and tested how it affects initiation, pausing, and termination by E. coli RNA polymerase. Recombinant GreA induced cleavage of two to three nucleotide fragments in two promoter-proximal complexes, whereas an apparently endogenous cleavage removed a single larger fragment. Both types of cleavage stopped once the transcript was shortened to approximately 10 nucleotides. However, during initiation, GreA induced cleavage of transcripts as short as four nucleotides, inhibiting their release as abortive products and stimulating both productive initiation and "primer-shifting" at a weak promoter. GreA induced repetitive cleavage over a long distance in complexes containing a long G-less nascent transcript. However, reverse translocation was inhibited in transcription complexes that contained a G-rich, C-less nascent transcript. Substituting IMP for GMP in the transcript relieved inhibition. Finally, GreA had little effect on transcription through the his and trp leader pause sites or on termination at nine different p-independent terminators. We propose that transcript cleavage and reverse translocation are controlled in part by backsliding of the nascent transcript through an RNA-binding site.

Bacterial Proteins↗