Biomedical subjects

R Landick

Publications and source records attributed to R Landick.

At least 55 records · Page 3Linked to original sources

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↗

PubMed1994

GreA-induced transcript cleavage is accompanied by reverse translocation to a different transcription complex conformation.

GreA- and GreB-induced transcript cleavage drives reverse translocation of Escherichia coli RNA polymerase on a DNA template in the absence of NTPs (Feng, G.-H., Lee, D. N., Wang, D., Chan, C. L., and Landick, R. (1994) J. Biol. Chem. 269, 22282-22294, accompanying report). During transcript elongation, the sizes of the DNA footprint and the single-stranded transcription bubble vary markedly among transcription complexes halted at different template positions. To test whether transcription complex intermediates formed during transcript cleavage-induced reverse translocation also display heterogeneous conformations at different template positions, we examined the structures of two different transcription complexes before and after GreA treatment. Transcription complexes halted at position +16 after initiation at the T7 A1 promoter or paused at the trpL pause site exhibited strong blocks to transcript cleavage after removal of 6 to 10 nucleotides. In both cases, the down-stream contact between RNA polymerase and DNA moved little during transcript cleavage, thereby increasing its distance from the active site, whereas the upstream DNA contact and the borders of the transcription bubble moved in approximate register with the transcript 3'-end. The backward movements of halted E. coli RNA polymerase are similar to a recently postulated model for discontinuous translocation during transcription, but differ from those reported for arrested RNA polymerase II transcription complexes.

Bacterial Proteins↗

PubMed1994

Tethered particle motion method for studying transcript elongation by a single RNA polymerase molecule.

Schafer et al. (Nature 352:444-448 (1991)) devised the tethered particle motion (TPM) method to detect directly the movement of single, isolated molecules of a processive nucleic acid polymerase along a template DNA molecule. In TPM studies, the polymerase molecule is immobilized on a glass surface, and a particle (e.g., a 0.23 microns diameter polystyrene bead) is attached to one end of the enzyme-bound DNA molecule. Time-resolved measurements of the DNA contour length between the particle and the immobilized enzyme (the "tether length") are made by determining the magnitude of the Brownian motion of the DNA-tethered particle using light microscopy and digital image processing. We report here improved sample preparation methods that permit TPM data collection on transcript elongation by the Escherichia coli RNA polymerase at rates (approximately 10(2)-fold higher than those previously obtained) sufficient for practical use of microscopic kinetics techniques to analyze polymerase reaction mechanisms. In earlier TPM experiments, calculation of tether length from the observed Brownian motion was based on an untested numerical simulation of tethered bead Brownian motion. Using the improved methods, we have now empirically validated the TPM technique for tether lengths of 308-1915 base pairs (bp) using calibration specimens containing particles tethered by individual DNA molecules of known lengths. TPM analysis of such specimens yielded a linear calibration curve relating observed Brownian motion to tether length and allowed determination of the accuracy of the technique and measurement of how temporal bandwidth, tether length, and other experimental variables affect measurement precision. Under a standard set of experimental conditions (0.23 microns diameter bead, 0.23 Hz bandwidth, 23 degrees), accuracy is 108 and 258 bp r.m.s. at tether lengths of 308 and 1915 bp, respectively. Precision improves linearly with decreasing tether length to an extrapolated instrumentation limit of 10 bp r.m.s. and improves proportionally to the inverse square root of measurement bandwidth (1.9 x 10(2) bp Hz-1/2 for 1090-bp tethers). Measurements on large numbers of individual polymerase molecules reveal that time-averaged single-molecule elongation rates are more variable than is predicted from the random error in TPM measurements, demonstrating that the surface-immobilized RNA polymerase molecules are kinetically heterogeneous.

Biophysical Phenomena↗

PubMed1993

Four contiguous amino acids define the target for streptolydigin resistance in the beta subunit of Escherichia coli RNA polymerase.

Streptolydigin (stl), a bacteriostatic inhibitor of transcription elongation, interacts with the beta subunit of Escherichia coli RNA polymerase. We have defined the target for stl resistance using chemical mutagenesis and mutagenic polymerase chain reaction. Mutations resulting in stl resistance are confined to a small cluster of contiguous amino acids, amino acids 543 to 546. These stlr mutants differ from one another in their levels of resistance to stl in vivo and in vitro. We have analyzed two of the mutants, A543V and F545S, for their effects on elongation and termination in vivo and in vitro. Neither affected termination at rho-dependent or rho-independent terminators. These mutants were indistinguishable from wild type in a T7 in vitro elongation assay. F545S, however, did exhibit slower elongation kinetics in a lambda tR1 pausing assay. We conclude that mutations in the stlr region can influence transcription elongation, but that these amino acids are not directly involved in catalysis.

Amino Acid Sequence↗

PubMed1993

Dissection of the his leader pause site by base substitution reveals a multipartite signal that includes a pause RNA hairpin.

A key feature of transcriptional attenuation in some amino acid biosynthetic operons is a transcriptional pause that occurs immediately after synthesis of the first leader transcript secondary structure. Both RNA secondary structure and downstream DNA sequence are important for pausing at these sites; however, the precise RNA structures involved and the relative contribution of other RNA and DNA bases to pausing are unknown. We studied the effects of base substitutions upstream from the his leader pause site (immediately prior to addition of G103) to determine how nucleic acid sequences and RNA structure contribute to pausing. By testing compensatory base substitutions, we found that pausing depended in part on an RNA secondary structure containing a five base-pair stem and eight nucleotide loop, which we call the his pause RNA hairpin. The his pause hairpin forms 11 nucleotides upstream from the paused transcript 3' end and thus corresponds to only the upper portion of the larger his A:B leader transcript secondary structure. Some base substitutions in the ten nucleotides between the pause hairpin and the 3' end of the transcript increased pausing, whereas others decreased pausing. However, compensatory substitutions that restored pairing of these bases in the lower portion of the A:B secondary structure did not alter these effects. Changing the 3'-terminal nucleotide of the transcript (U102) altered both the position and strength of pausing. Thus, in addition to the downstream DNA sequence, three distinct segments of nucleic acid upstream from the nucleotide-addition site in the transcription complex contribute to pausing in different ways: the pause RNA hairpin, the 3'-proximal region of transcript or DNA template, and the 3'-terminal nucleotide. We suggest that electrostatic interaction between the pause hairpin and RNA polymerase, rather than disruption of an RNA:DNA heteroduplex, delays elongation at the his leader pause site.

Bacterial Proteins↗

PubMed1992

Structure of RNA and DNA chains in paused transcription complexes containing Escherichia coli RNA polymerase.

RNA polymerases pause conspicuously at certain positions on a DNA template. At the well-studied pause sites in the attenuation control regions that precede the trp and his operons, both formation of secondary structure in the nascent transcript and the DNA sequence immediately downstream contribute to pausing. The mechanisms of these effects are unknown. We report here studies on the structure of the RNA and DNA strands in purified trp and his paused transcription complexes in comparison to ten elongation complexes halted by nucleoside triphosphate deprivation. A 14 to 22 nucleotide region of the DNA strands was accessible to modification by KMnO4 or diethylpyrocarbonate in both the paused and halted transcription complexes. However, the region in front of the nucleotide-addition site was reactive only in some halted complexes. In both types of complexes, approximately eight nucleotides on the template strand immediately preceding the 3' end were protected from modification. We also examined the sensitivity of the nascent transcript to RNase A and found that the 3'-proximal eight nucleotide region could be cleaved without complete loss of the potential for elongation. However, a model RNA:DNA hybrid designed to mimic a hybrid in the transcription complex could also be cleaved under similar conditions. Together, the results suggest that the 3'-proximal eight nucleotides of transcript may pair with the DNA template and that this structure is not disrupted by hairpin formation at a pause site. Rather, pausing may result from distinct interactions between RNA polymerase and both the pause RNA hairpin and the downstream DNA sequence.

Base Sequence↗

PubMed1991

Transcription by single molecules of RNA polymerase observed by light microscopy.

The kinetics of transcription by Escherichia coli RNA polymerase relate directly to the regulation of transcription and to the properties of processive enzymes in general, but analysis of RNA polymerase movement along the DNA template has so far been limited to the study of populations of enzyme molecules. The ability to view nanometre-sized particles with the light microscope suggested a method of monitoring transcription by individual RNA polymerase molecules. We describe here the behaviour of 40-nm-diameter particles of colloidal gold attached to the ends of DNA molecules being transcribed by RNA polymerase immobilized on a glass surface. The tethered gold particles are released from the surface at times after addition of nucleoside triphosphates that are consistent with the kinetics of transcription by RNA polymerase in solution. Analysis of the brownian motion of the gold particles enabled us to measure the movement along the template DNA of individual polymerase molecules.

Bacterial Proteins↗

PubMed1991

Analysis of premature termination in c-myc during transcription by RNA polymerase II in a HeLa nuclear extract.

Transcriptional regulation of the human c-myc gene, an important aspect of cellular differentiation, occurs in part at the level of transcript elongation. In vivo, transcriptional arrest, due to either pausing or termination, occurs near the junction between the first exon and first intron and varies with the growth state of the cell. We have tested the transcription of c-myc templates in HeLa nuclear extracts. We did not observe significant arrest under standard conditions, but we found that a considerable fraction of transcription complexes stopped at the c-myc TII site (just past the first exon-intron junction) when the KCl concentration was raised to 400 mM during elongation. Transcriptional arrest at TII also was observed at KCl concentrations as low as 130 mM and when potassium acetate or potassium glutamate was substituted for KCl. Under these conditions, arrest occurred at the TII site when transcription was initiated at either the c-myc P2 promoter or the adenovirus 2 major late promoter. Further, the TII sequence itself, in forward but not reverse orientation, was sufficient to stop transcription in a HeLa nuclear extract. By separating the TII RNA from active transcription complexes by using gel filtration, we found that arrest at TII at 400 mM KCl resulted in transcript release and thus true transcriptional termination. The efficiency of termination at TII depended on the growth state of the cells from which the extracts were made, suggesting that some factor or factors control premature termination in c-myc.

Acetates↗

PubMed1990

Replacement of the Escherichia coli trp operon attenuation control codons alters operon expression.

To test features of the current model of transcription attenuation in amino acid biosynthetic operons, alterations were introduced into the trp operon leader region and expression of the mutated operons was examined in miaA and miaA+ Escherichia coli strains that lacked the trp repressor. The miaA mutation prevents modification of the adenosine residue immediately 3' of the anticodon of tRNAs that interact with codons beginning with uridine. The undermodified tRNA(Trp) in miaA strains is thought to increase readthrough at the trp attenuator by slowing ribosome movement over two tandem Trp codons in the 14-codon leader peptide coding region. The rate of translation of these two "control codons" is thought to be the key step in determining the extent of transcription attenuation in the trp leader region. Sequential deletion of trpL DNA specifying the leader peptide initiation region, RNA segment 1, RNA segment 2 and RNA segment 3 alternately decreased and increased trp operon expression, a result consistent with previous findings in another bacterium and the generally accepted model for transcription attenuation. Replacement of the tandem Trp control codons by AGG-UGC (Arg-Cys) codons eliminated the miaA-dependent increase in transcription readthrough. Replacement of the Trp control codons by AGG-UGA (Arg-stop) codons caused complete readthrough at the trp attenuator as well as abolishing the miaA effect. Presumably, the ribosome terminating translation at the new UGA codon mimics the effect of a stalled ribosome at the Trp control codons. This finding suggests that ribosome dissociation at some stop codons is slow relative to the time required for transcription of the trp leader region. Thus, most ribosomes translating the trp leader peptide coding region may remain attached to the natural UGA stop codon until after the attenuation decision is made. The interpretation supports models for trp operon attenuation in which the elevated basal level readthrough is determined by occasional ribosome release prior to synthesis of the 3:4 terminator hairpin.

Amino Acid Sequence↗

PubMed1990

Transcription pausing by Escherichia coli RNA polymerase is modulated by downstream DNA sequences.

Escherichia coli RNA polymerase pauses immediately after transcription of certain sequences that can form stable secondary structures in the nascent RNA transcript; pausing appears to be essential for several types of bacterial transcription attenuation mechanisms. Because base changes that weaken the RNA secondary structures reduce the half-life of pausing by RNA polymerase, nascent transcript RNA hairpins are thought to cause pausing at these sites. We show here that, for the well characterized trpL pause site, the determinants of transcription pausing are not limited to the RNA hairpin, but include the not-yet-transcribed sequence of DNA immediately downstream from the pause site. We show that this effect extends to bases up to fourteen nucleotides downstream from the pause site, that placement of a oligo(dT) tract in the nontranscribed strand in this region does not convert the pause site to a termination site, and that shifting the position of pausing by one nucleotide downstream almost eliminates pausing. From an analysis of many variants of this downstream sequence, we argue that the effect of downstream sequence is not related simply to its GC content. We suggest that these effects are mediated by altered interactions between RNA polymerase and the DNA template downstream from the enzyme's active site.

Base Sequence↗

PubMed1990

Nucleotide sequence and genetic characterization reveal six essential genes for the LIV-I and LS transport systems of Escherichia coli.

The nucleotide sequence of the genes encoding the high affinity, branched-chain amino acid transport systems LIV-I and LS has been determined. Seven genes are present on a 7568-base pair DNA fragment, six of which participate directly in branched-chain amino acid transport. Two periplasmic amino acid-binding proteins are encoded by the livJ (LIV-BP) and livK (LS-BP) genes. These two proteins confer specificity on the LIV-I and LS transport systems. livK is the first gene in a polycistronic message that includes four genes encoding membrane components, livHMGF. The protein products of the livHMGF genes are shared by the two systems. An analysis of the livH and livM DNA sequences suggests that they encode hydrophobic proteins capable of spanning the membrane several times. The LivG and LivF proteins are less hydrophobic, but are also tightly associated with the membrane. Both LivG and LivF contain the consensus sequence for adenine nucleotide binding observed in many other transport proteins. A deletion strain that does not express any of the liv genes was constructed. This strain was used to show that each of the membrane component genes is required for high affinity leucine transport, including two genes, livM and livF, for which no previous genetic evidence had been obtained.

Amino Acid Sequence↗

PubMed1990

Amino acid changes in conserved regions of the beta-subunit of Escherichia coli RNA polymerase alter transcription pausing and termination.

Control of transcription at pause and termination sites is common in bacteria. Many transcriptional pause and termination events are thought to occur in response to formation of an RNA hairpin in the nascent transcript. Some mutations in the beta-subunit of Escherichia coli RNA polymerase that confer resistance to the transcription inhibitor rifampicin also alter the response to transcriptional pause and termination signals. Here, we report isolation of termination-altering mutations that do not confer rifampicin resistance and show that such mutations occur predominantly in limited regions of the beta-subunit polypeptide. One region is between amino acid residues 500 and 575, which encompasses the locations of almost all known rifampicin-resistance mutations. Many termination-altering mutations also occur in two other regions: between amino acid residues 740 and 840 and near the carboxyl terminus of the beta-subunit (amino acid residues 1225-1342). Amino acid sequences in these three regions of the beta-subunit are conserved between prokaryotic and eukaryotic beta-subunit homologs. Several mutations that alter transcription termination in vitro affect amino acid residues that are identical in prokaryotic and eukaryotic RNA polymerase beta-subunit homologs, suggesting that they alter an important function common to multisubunit RNA polymerases. We propose that these three regions of the beta-subunit may contact the nascent RNA transcript, the RNA-DNA heteroduplex, or the DNA template in the transcription complex and that mutations in these regions alter transcription pausing and termination by affecting these contacts.

Amino Acid Sequence↗

PubMed1990

Insertional mutagenesis of a plasmid-borne Escherichia coli rpoB gene reveals alterations that inhibit beta-subunit assembly into RNA polymerase.

A plasmid was constructed that overproduces the Escherichia coli RNA polymerase beta subunit from a lac promoter-rpoB fusion. The overproduced, plasmid-encoded beta subunit assembled into functional RNA polymerase that supplied greater than 90% of the transcriptional capacity of the cells. Excess beta subunit segregated into insoluble inclusion bodies and was not deleterious to cell growth. By insertion of a XhoI linker sequence (CTCGAG) and accompanying deletion of variable amounts of rpoB sequences, 13 structural alterations were isolated in the first and last thirds of the plasmid-borne rpoB gene. Twelve of these alterations appeared to reduce or prevent assembly of plasmid-encoded beta subunit into RNA polymerase. One alteration had no discernible effect on assembly or function of the beta subunit; eight others appeared to inhibit assembly but still produced detectable transcriptional activity. Three of these nine alterations produced beta-subunit polypeptides that inhibited cell growth at 32 degrees C, even though they were present in less than 50% of the cell RNA polymerase. When assembled into RNA polymerase, these three altered beta subunits apparently affected essential RNA polymerase functions. Four of the recovered alterations appeared to inhibit completely or almost completely assembly of the beta subunit into RNA polymerase. The results are consistent with a hypothesis that sequences in the first third of the beta-subunit polypeptide are especially important for proper folding and assembly of the beta subunit.

Base Sequence↗

PubMed1989

The Salmonella typhimurium his operon leader region contains an RNA hairpin-dependent transcription pause site. Mechanistic implications of the effect on pausing of altered RNA hairpins.

The Salmonella typhimurium his leader region contains a well documented transcription attenuator. We report here the results of in vitro transcription studies that characterized a transcription pause site in the his leader region. The pause occurred after synthesis of the first his leader secondary structure (A:B) and immediately preceding addition of G103 to the nascent transcript. RNA polymerase pausing at this site would allow a ribosome synthesizing the his leader peptide to release the paused polymerase and synchronize transcription and translation of the his leader region. The half-life of transcription complexes paused in the his leader was enhanced by NusA, but not guanosine 5'-diphosphate 3'-diphosphate. Nuclease digestion and RNA modeling studies were consistent with a compact three-dimensional structure for the his pause RNA. The half-life of the his leader paused transcription complex was decreased greatly on altered templates in which the C71-G93 base pair was disrupted but was unchanged when the C65-G100 base pair was disrupted. This result is consistent with a model for the structure of paused transcription complexes in which a portion of the RNA:DNA elongation heteroduplex is retained.

Base Sequence↗

PubMed1987

Isolation and structural analysis of the Escherichia coli trp leader paused transcription complex.

Transcription pausing is a key step in many prokaryotic transcription attenuation mechanisms. Pausing is thought to occur when an RNA hairpin forms near the 3' end of a growing transcript. We report here the isolation of the trp leader paused transcription complex containing a defined 92-nucleotide nascent transcript. Digestion of isolated paused complexes with RNase T1 suggests that the trp leader RNA hairpin designated 1:2 forms in the paused transcription complex. The transcription factor NusA alters the RNase T1 digestion pattern of the 92-nucleotide pause transcript in the complex but not the cleavage patterns of purified pause RNA, suggesting that NusA specifically affects the 1:2 hairpin in the paused transcription complex. The isolated paused transcription complex retains the ability to resume transcription. Kinetic studies on the resumption of elongation suggest that NusA is a non-competitive inhibitor of paused complex release and that the Ks for GTP is around 300 microM. RNA polymerase in the paused transcription complex protects approximately 30 base-pairs on both DNA strands from exonuclease digestion.

Bacterial Proteins↗

PubMed1987

Detection of transcription-pausing in vivo in the trp operon leader region.

To determine whether RNA polymerase pauses during transcription in vivo, we have examined transcripts of the trp operon leader regions of Serratia marcescens and Escherichia coli. Labeled RNAs synthesized in E. coli strains containing plasmids bearing wild-type or mutant trp leader regions of S. marcescens or E. coli were isolated by hybridization and analyzed by polyacrylamide gel electrophoresis. The labeled RNAs synthesized in vivo on the S. marcescens wild-type and deletion mutant plasmids were the same size as the in vitro pause and leader transcripts. Hybridization of the presumed in vivo pause RNAs, and control in vitro pause RNAs, to M13 phage DNA containing a trp leader region deletion followed by treatment with S1 nuclease produced identical protected RNA species, proving that the in vitro and in vivo RNAs were identical. The amount of labeled pause RNAs relative to leader RNAs decreased following a chase with unlabeled uridine. E. coli RNAs identical to the previously characterized in vitro pause and leader transcripts were demonstrated by electrophoretic band position and fingerprint analysis. The finding that transcription pausing occurs in vivo is consistent with the view that transcription pausing and ribosome release of paused transcription complexes are responsible for the coupling of translation with transcription that is crucial to attenuation.

Escherichia coli↗