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D S Luse

Publications and source records attributed to D S Luse.

36 records · Page 2Linked to original sources
PubMed1992

The RNA polymerase II ternary complex cleaves the nascent transcript in a 3'----5' direction in the presence of elongation factor SII.

The process by which RNA polymerase II elongates RNA chains remains poorly understood. Elongation factor SII is known to be required to maximize readthrough at intrinsic termination sites in vitro. We found that SII has the additional and unanticipated property of facilitating transcript cleavage by the ternary complex. We first noticed that the addition of SII caused a shortening of transcripts generated by RNA polymerase II at intrinsic termination sites during transcription reactions in which a single NTP was limiting. Truncation of the nascent transcript was subsequently observed using a series of ternary complexes artificially paused after the synthesis of 15-, 18-, 20-, 21-, and 35-nucleotide transcripts. Transcripts as short as 9 or 10 nucleotides were generated in 5-min reactions. All of these shortened RNAs remained in active ternary complexes because they could be chased quantitatively. Continuation of the truncation reaction produced RNAs as short as 4 nucleotides; however, once cleavage had proceeded to within 8 or 9 bases of the 5' end, the resulting transcription complexes could not elongate the RNAs with NTP addition. Transcript cleavage requires a divalent cation, appears to proceed primarily in 2-nucleotide increments, and is inhibited by alpha-amanitin. The catalytic site of RNA polymerase II is repositioned after transcript cleavage such that polymerization resumes at the proper location on the template strand. The extent and kinetics of the transcript truncation reaction are affected by both the position at which RNA polymerase is halted and the sequence of the transcript.

Animals↗
PubMed1991

Abortive initiation is increased only for the weakest members of a set of down mutants of the adenovirus 2 major late promoter.

We have shown that accurate initiation of productive RNA synthesis in vitro at the adenovirus 2 major late promoter is accompanied by abortive initiation of very short transcripts (Luse, D. S., and Jacob, G. A. (1987) J. Biol. Chem. 262, 14990-14997). We made a set of sequence variants of this promoter, using every possible base at position -28 (in the TATA box) in the context of either the normal base (A) or a T at position +1 on the nontemplate strand. All changes from wild type reduced promoter strength. The two weakest promoters were 10- and 30-fold less active than wild type in productive RNA synthesis. We tested the possibility that the down mutations also caused an increase in the proportion of in vitro initiations which are abortive. This effect was seen only with the two weakest members of the promoter set. For these promoters, which share an A----C change at the -28 position of the TATA box, the ratio of abortive to productive initiations was 3-4-fold higher than for the other promoters. Interestingly, the sequence change at +1, although a down mutation, did not lead to an increase in abortive initiation.

Adenoviruses, Human↗
PubMed1991

Transcription on nucleosomal templates by RNA polymerase II in vitro: inhibition of elongation with enhancement of sequence-specific pausing.

The process by which RNA polymerase II elongates RNA chains in vivo, where the template is at least partially in a nucleosomal configuration, remains poorly understood. To approach this question we have partially purified RNA polymerase II transcription complexes paused early in elongation. These complexes were then used as substrates for chromatin reconstitution. Elongation of the nascent RNA chains on these nucleosomal templates is severely inhibited relative to elongation on naked DNA templates. Elongation on the nucleosomal templates results in a reproducible template-specific pattern of transcripts generated by RNA polymerase pausing. The RNA polymerases are not terminated because the large majority will resume elongation upon the addition of Sarkosyl or 400 mM KCl. The effectiveness of RNA polymerase II pause/termination sites is enhanced by the presence of nucleosomes. For example, a pause site similar in sequence to the c-myc gene exon 1 terminator is used four to seven times more effectively in reconstituted templates. A comparison of elongation on templates bearing phased nucleosomes and on reconstituted templates that show no predominant phasing pattern indicates that the locations of pause sites are not related to the positions of the nucleosomes. Rather, the major determinant of RNA polymerase pausing on the nucleosomal templates appears to be the underlying DNA sequence.

Adenoviruses, Human↗
PubMed1991

RNA polymerase II elongation complexes paused after the synthesis of 15- or 35-base transcripts have different structures.

We have purified specific RNA polymerase II elongation intermediates initiated at the adenovirus type 2 major late promoter and paused either 15 or 35 to 36 bases downstream of the transcription initiation site. Transcription was arrested at these two sites by combining modification of the promoter sequence with limitation of appropriate nucleotide concentrations in the in vitro reaction. The resultant complexes were remarkably stable and could be purified away from free DNA and contaminating protein-DNA complexes, without loss of activity, by the use of sucrose gradient sedimentation and low-ionic-strength polyacrylamide gel electrophoresis. The complexes were characterized by both DNase I and o-phenanthroline-copper ion nuclease protection assays. The DNase I footprints revealed that the structures of the 15- and 35- to 36-nucleotide transcription complexes differed from those previously reported for an adenovirus type 2 major late preinitiation complex and a subsequent intermediate formed upon addition of ATP. Furthermore, the 35- to 36-nucleotide complex protected a significantly smaller portion of the template than the 15-nucleotide species and migrated at a slightly higher rate in polyacrylamide gels. These observations suggest that changes in structural organization may continue to occur in transcription complexes which are already committed to elongation.

Base Sequence↗
PubMed1988

Assembly of RNA polymerase II preinitiation complexes before assembly of nucleosomes allows efficient initiation of transcription on nucleosomal templates.

We have previously shown that assembly of nucleosomes on the DNA template blocks transcription initiation by RNA polymerase II in vitro. In the studies reported here, we demonstrate that assembly of a complete RNA polymerase II preinitiation complex before nucleosome assembly results in nucleosomal templates which support initiation in vitro as efficiently as naked DNA. Control experiments prove that our observations are not the result of slow displacement of nucleosomes by the transcription machinery during chromatin assembly, nor are they an artifact of inefficient nucleosome deposition on templates already bearing an RNA polymerase. Thus, the RNA polymerase II preinitiation complex appears to be resistant to disruption by subsequent nucleosome assembly.

Chromatin↗
PubMed1987

Abortive initiation by RNA polymerase II in vitro at the adenovirus 2 major late promoter.

We have investigated the formation of the first phosphodiester bond by RNA polymerase II in vitro. The template was a cloned DNA bearing the adenovirus 2 major late promoter; transcription factors and RNA polymerase II were provided by a HeLa cell nuclear extract. Dinucleotide primers and single nucleoside triphosphates were used as substrates. We found that accurate initiation does occur when only one phosphodiester bond can be formed; however, all of the resulting dinucleotide-primed trimers are abortively initiated. Synthesis of the trimers by RNA polymerase II requires ATP or dATP and is sensitive to low concentrations of alpha-amanitin. Treatments which abolish the ability of the preinitiation complex to synthesize long RNAs also eliminate the ability to abortively initiate. Abortive initiation proceeds for at least one-half h at 25 degrees C, at which point up to 4 mol of transcript/mol of template have been synthesized. The level of abortive initiation (per template molecule) is not significantly reduced by 0.025% Sarkosyl or by 10-fold dilution of the reaction, consistent with the initiation complex remaining intact during abortive initiation.

Adenoviruses, Human↗
PubMed1987

Transcription initiation by RNA polymerase II in vitro. At least two nucleotides must be added to form a stable ternary complex.

We have prepared RNA polymerase II preinitiation complexes by incubating templates containing the adenovirus 2 major late promoter with HeLa cell nuclear extracts in the absence of nucleoside triphosphates. These preinitiation complexes are partially purified by gel filtration and are then provided with the appropriate substrates to allow either one or two phosphodiester bonds to be formed. When substrates that allow only one bond to form are used, no stable ternary complex is obtained and no RNA is made that can be incorporated into longer RNA chains. A stable complex is obtained, however, if the RNA polymerase can make two bonds. The production of a stable ternary complex requires ATP or dATP and is inhibited by alpha-amanitin. In the course of exploring the energy requirement for initiation we found that dATP may be incorporated, in the absence of ATP, as the initial base of the RNA. However, deoxyribonucleotides are not appreciably incorporated into the body of the transcript after the first two bases have been added to the growing chain.

Adenosine Triphosphate↗
PubMed1987

Transcription initiation by RNA polymerase II in vitro. Properties of preinitiation, initiation, and elongation complexes.

We have prepared three types of RNA polymerase II transcription complexes: a preinitiation complex (complex 0), a complex which has synthesized two phosphodiester bonds (complex 2), and a complex which has synthesized 10-13 bonds (complex 10). We have studied the differential response of these complexes to a variety of disruptions: detergent (Sarkosyl), high levels of KCl, extended incubation at 25 degrees C, proteolysis, and digestion with DNase I. Complex 0 is extremely stable at 25 degrees C in the absence of ATP, but it is sensitive to the other treatments including 25 degrees C incubation in the presence of ATP. Once the complex has made two phosphodiester bonds, the properties almost reverse from those of complex 0; complex 2 remains unstable at 25 degrees C in the presence of ATP but is resistant to high levels of Sarkosyl and KCl, to extensive DNase I digestion, and to brief proteolysis. Addition of 10 or more bases to the growing RNA chain results in a complex completely resistant to all of the treatments used. When DNase I-trimmed complex 0 is allowed to initiate RNA synthesis, chains of about 33 bases are obtained. In contrast, DNase-trimmed complex 2 gives only about 23 base transcripts; DNase-treated complex 10 will elongate its nascent chains by about 21 bases as well (to give, on average, 34 base transcripts).

Adenosine Triphosphate↗
PubMed1987

Variations in template protection by the RNA polymerase II transcription complex during the initiation process.

Preinitiation complexes (complex 0) or complexes which either made 2 or an average of 10 phosphodiester bonds (complexes 2 and 10, respectively) were assembled in vitro on the adenovirus 2 major late promoter. Each of the complexes was digested extensively with DNase I; the protected DNAs were purified and hybridized in a series of end-labeled oligonucleotides homologous to sequences on the coding or noncoding strands near the initiation site. The hybrids were then extended with reverse transcriptase to map the extent of template protection conferred by proteins in the complex. The downstream protection edge revealed by this approach was approximately +30, +25, and +35 for complexes 0, 2, and 10, respectively. We subsequently found that the apparent inward movement of the downstream protection boundary on initiation could be produced by satisfying the energy requirement for transcription initiation (i.e., by treating with ATP or dATP). The downstream boundary change occurred as rapidly as we could perform the test (less than 60 s) and was not blocked by alpha-amanitin. DNAs from trimmed complexes 0, 2, or 10 all supported extension to a single upstream edge at about position -42. Upstream protection was stable in the preinitiation complex, but when postinitiation complexes were incubated for extended periods, protection of the entire upstream region was lost. This decay of upstream protection, like the movement of the downstream boundary, was found to result from exposure to ATP or dATP. Unlike the downstream boundary movement, however, the upstream change was relatively slow; about 15 min was required to lose one-half of the protection.

Adenosine Triphosphate↗
PubMed1986

The presence of nucleosomes on a DNA template prevents initiation by RNA polymerase II in vitro.

RNA was synthesized in vitro using HeLa cell nuclear extracts and circular DNA templates onto which varying numbers of nucleosomes had been reconstituted with Xenopus oocyte extracts. We found that fully reconstituted templates supported no specific initiation by RNA polymerase II; however, DNA exposed to the reconstitution extracts under conditions which did not allow nucleosome deposition was transcribed normally. A set of successively less reconstituted templates was also transcribed. No initiation occurred on reconstitutes with more than two-thirds of the physiological nucleosome density; reconstitutes with less than one-third of the physiological nucleosome density were transcribed as efficiently as naked DNA.

Adenoviruses, Human↗
PubMed1986

Differential expression of mouse beta/goat beta c, mouse beta/goat beta F, and mouse beta/goat epsilon II hybrid globin genes in murine erythroleukemia cells.

We assembled three hybrid beta-globin genes by fusing the mouse beta-major promoter and initial transcribed region to one of three goat beta-like globin gene bodies: beta c (preadult), beta F (fetal), or epsilon II (embryonic). Thymidine kinase (tk)-deficient murine erythroleukemia (MEL) cells were cotransformed with one of these constructs and a separate plasmid bearing the tk gene. Half of the 24 cell lines containing either the mouse beta/goat beta c or mouse beta/goat beta F genes expressed the transferred genes at significant levels; in many cases the hybrid genes were, like the endogenous beta-globin genes, inducible with dimethyl sulfoxide. We obtained 13 cell lines containing the mouse beta/goat epsilon II hybrid gene, 6 of which were cotransfected with a mouse beta/human beta fusion gene known to function in MEL cells. In contrast to the results with the other fusion genes, the mouse beta/goat epsilon II hybrid was very poorly expressed: in two separate experiments, 0 of 13 and 2 of 13 lines showed significant mouse beta/goat epsilon II RNA levels after induction. In all these lines the endogenous mouse beta and cotransfected mouse beta/human beta genes were expressed. As an initial test of possible reasons for the inactivity of the mouse beta/goat epsilon II hybrid, we recloned this fusion gene into a tk-bearing plasmid, adjacent to the tk gene. Of 12 cell lines transformed with this plasmid, 11 produced mouse beta/goat epsilon II RNA; in 6 cases the expression was both strong and dimethyl sulfoxide inducible.

Animals↗
PubMed1984

Purification and characterization of ternary complexes containing accurately initiated RNA polymerase II and less than 20 nucleotides of RNA.

We have previously demonstrated that transcription of the adenovirus type 2 (Ad2) late promoter in vitro under UTP-limiting conditions results in pauses by the elongating RNA polymerase II between positions +6 and +17. We report here the purification of complexes between the paused RNA polymerase and a 260 base-pair Ad2 promoter-bearing DNA fragment. The procedure involves sedimentation through sucrose gradients, electrophoresis in agarose gels, and electroelution from the gels; the final complex pool is completely active in chain elongation. We observe a sharp discontinuity in complex stability during purification as a function of the number of bases added to the growing chains: complexes in which the polymerase has added more than ten bases are stable and are active in chain elongation even after the electroelution step, whereas complexes containing seven or fewer bases dissociate very easily. When purified complexes are extensively digested with proteinase K their electrophoretic mobility is increased considerably, yet they remain fully active in chain elongation. If purified complexes are digested with DNase I their electrophoretic mobility does not change. When the nuclease-treated complexes are allowed to continue chain elongation, they are able to add approximately 20 more bases to the nascent chains.

Adenoviruses, Human↗
PubMed1983

Promoter-proximal pausing by RNA polymerase II in vitro: transcripts shorter than 20 nucleotides are not capped.

We have synthesized RNA from cloned adenovirus 2 late promoter DNA in an in vitro transcription extract under UTP-limiting conditions. Under these circumstances, almost all of the alpha-amanitin-sensitive RNA produced is shorter than 20 nucleotides; most of these short transcripts are present in four species, 6, 7, 13, and 17 nucleotides. These short RNAs are initiated at the adenovirus 2 promoter, as judged by partial sequence analysis and by the abolition of their synthesis upon cleavage of the template DNA at sites which also abolish the production of full-length transcripts. All of the short transcripts can be chased, with excess UTP, into 197-base run-off transcripts; thus, these RNAs are precursors of full-length transcripts and not synthetic "dead ends." Significantly, none of these short RNAs is capped or 2'-O-methylated. However, 79-base run-off transcripts synthesized from this promoter with nonlimiting NTP levels are fully capped.

Base Sequence↗
PubMed1981

Transcription of the beta-like globin genes and pseudogenes of the goat in a cell-free system.

We have examined the transcription in a cell-free system of all the members of a highly regulated gene family, the beta-like hemoglobin genes of the goat. These five genes, which code for embryonic, fetal, juvenile, and adult beta-globin proteins, are all transcribed to roughly the same extent in the in vitro system. In all cases initiation of transcription is accurate. However, two goat beta-globin pseudogenes, as well as several artificially constructed deletion mutants, are not transcribed in vitro. A common feature of the transcriptionally inactive genes is the lack of an AT-rich consensus sequence just upstream of the presumptive initiation site.

Animals↗
PubMed1979

Selective and accurate initiation of transcription at the Ad2 major late promotor in a soluble system dependent on purified RNA polymerase II and DNA.

Transcription of Ad2 DNA templates in the presence of crude cellular extracts supplemented with exogenous (purified) RNA polymerase II is selectively and accurately initiated at the major late viral promoter at map position 16.45. Specific initiation has been demonstrated by a combination of hybridization, nuclease S1 mapping, size and partial sequence (fingerprint) analyses of the transcripts generated with various templates. With intact Ad2 DNA, transcription is terminated ell before the end of the 28 kb transcription unit is reached. With truncated templates (which contain intact promoter regions and several hundred base pair segments of the transcribed region) the expected run-off products are observed, along with a low level of prematurely terminated transcripts. The 560 nucleotide run-off product of the Sma l-f template (coordinates 11.6-18.2) was shown to contain all the large RNAase T1 oligonuc eotides that are characteristic of the corresponding in vivo transcript from this region; in addition, the 5 terminal undecanucleotide appears to be both capped and methylated. We have investigated various parameters (salt, metal ion and template concentrations) that affect the level of specific transcription in the crude system and have found that, under optimal conditions, specific transcription of Ad2 DNA continues for several hours. In addition, specific transcription initiation at the late promoter is observed with extracts derived from either virus-infected or uninfected KB cells and with class II RNA polymerases isolated from either human calf, murine or amphibian cells. RNA polymerase II from wheat germ does not function in this system.

Adenoviruses, Human↗