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Effects of high mobility group proteins 1 and 2 on initiation and elongation of specific transcription by RNA polymerase II in vitro

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Effects of high mobility group proteins 1 and 2 on initiation and elongation of specific transcription by RNA polymerase II in vitro
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  Volume 16 Number 231988 Nucleic Acids Research Effects of high mobility group proteins 1 and 2on initiation and elongationof specific transcription by RNA polymerase H in vitro DavidJ.Tremethick+ and Peter L.Molloy* CSIRO Division of Biotechnology, Laboratory for Molecular Biology, NorthRyde, PO Box 184, NSW 2113, Australia ReceivedAugust 31, 1988; Revised and Accepted November 3, 1988 ABSTRACT High mobility group proteins 1 and 2 (HMGs 1 and 2) are abundant chromosomal proteins of higher eukaryotes, which havebeen found to be enriched in regions of active chromatin. We have previously demonstrated that they can stimulate specific transcription invitro by RNA polymerases II and Ill andovercome inhibition caused by added histones. Here we study whether these effects are mediated at the level of initiation or elongation of transcription. Additions of HMGs 1 and 2 and/or histones werefound to have only smallor no effect on theefficiency of elongation; this was determined bycomparing the relative synthesis of transcripts of differentlengths, ranging from 95 to 1535 bases. The observed stimulation cannot be explained by an increased utilization of initiation complexes for multiple roundsof transcription as a similar level of stimulation by HMGs 1 and 2 was seen when RNA synthesis was limited to oneround per template DNA by addition ofa low level of Sarkosyl after formation of initiation complexes. The effects of HMGs 1 and2were principally seen on the rate of formation of effective initiation complexes. These data areconsistent with the hypothesis that HMGs 1 and 2 stimulate transcription by facilitating the formation of active initiation complexes on template DNA. INTRODUCTION The role of chromosomal proteins andnucleosome structure in modulation of transcriptional activityin eukaryotes is still far from clear. Nuclease digestion studies have indicated that the structure of active chromatin is more open oraccessible when compared to the structure of inactive chromatin  1,2). This suggests that a major function of chromatin structure is either to allow or prevent the access of transcription factors and/or RNA polymerases to promoter and transcribed regions. A number of studies have shown that while histones are associated with activelytranscribed DNA in vivothere is considerable disruption of the normalnucleosomal organization  3-10). Otherchanges which havebeen correlated with regions of active chromatin include the presence of reduced levels of histone Hi and the presence of non-histone proteins  especiallythe high mobility group proteins), modified histones  e.g. acetylated or ubiquitinated) and modified nucleosome structure  11). High mobility group proteins 1 and2 (HMGs 1 and 2) are abundant chromosomal proteins of higher eukaryotes initially characterized by their unusual physicalproperties  12). A number of studies have provided evidence that they are preferentiallyassociated with 11107  Nucleic AcidsResearch active regions of chromatin  13-16) and it has been suggested that they are associated with the nucleosomal linkerregion. Using invitro transcription as a functional assay we demonstrated that HMGs 1 and 2 could stimulate specific and accurate transcription by RNA polymerases II and III  17), andovercome inhibition caused by addition of total histones to such reactions. We have recently examined the effects of HMGs 1 and 2on the binding to DNA of a sequence specifictranscription factor (MLTF) required for optimal transcription from the adenovirus major late promoter (AdMLP) andfound that HMGs 1 and 2 increase the rate offormation of DNA-MLTF complexes and also alter the DNaseI footprint pattern of MLTF  18). Here we have used functional transcrption assays to determine whether stimulation of transcription invitro by HMGs 1 and 2 occurs at the initiation steps of transcription or during chain elongation  orboth). Recent studies of specifictranscription by RNA polymerase 11 using the adenovirus major later promoterhavedemonstrated that multiple steps and at least four general 19-27) and one specific transcription factor  25,28,29) are involved in the formation of an activelytranscribing RNA polymerase II complex  see Figure 1). These steps include the binding of transcription factors to promoter sequences inthe DNA  template commitment or pre- initiation complex formation) followed by binding of RNA polymerase II and furtherfactors to form an initiation complex which is capable of initiating transcription upon addition of nucleoside triphosphates. Using low levels of the anionic detergent Sarkosyl it is possible to inhibit step II, the conversion of pre-initiation or template-committed complexes to competent initiation complexes  19,21) without inhibiting the conversion of initiation complexes to elongating RNA polymerase II complexes. For subsequent rounds of transcription from the same DNA template the Sarkosyl-sensitive step ofconversion of a pre-initiation complex to a competent initiation complex must be passed through again. In whole cell extracts it is possibletherefore to distinguish effects on initiation complex formation, their subsequent re- use forfurther rounds of transcription and elongation of RNA transcripts. MATERIALS AND METHODS Preparation of HMGs 1 and 2 and Histones: HMGs 1 and 2were extracted and fractionated from calf liver or thymus, and histones purified from mouse Ehrlich ascites cells as previously described  17). DNA Templates: Plasmid DNAs were isolated and prepared fortranscription reactions as described  17). Plasmid pHIIB  30) cut with SmaI gives a run-off RNA transcript from the adenovirus major late promoter of 536 bases. Plasmid pAdMLP was constructed from pHHIB by digestion with HindH and religation. It thuscontains the adenovirus type 2 major late promoter region from base -61 to base 192 relative to the major late transcription start site inserted at the HindIlI site of pBR322 and in the same numerical orientation. Plasmids pML(C2AT)lg and p(C2AT)19 were kindly provided byDrs Sawadogo 11108  Nucleic AcidsResearch DNA   < TFIIA,  AB], STF e- TFIID, D], BTF1 PRE- INITIATION COMPLEX r-RNA POLYMERASE 11 2 BF [TFIIB cB] BTF3 RAPs INITIATION -COMPETENT COMPLEX 3 t .-NTPs INITIATED COMPLEX Figure 1. Essential steps in transcription by RNA polymerase II. The scheme is drawn largely from the work of thelaboratories of Drs. R. Roeder P. Sharp and P. Chambon  19- 24). Transcription factor or fraction designations TFIIA, B, D and E, [AB],[DB], [CB], or STF, BTF1, 2 and 3 are those used respectively by thedifferent laboratories and factors which are probably equivalent are aligned. For the adenovirus major late promoter an additional promoter-specific transcription factor (USF, MLTF or Ad2MLP-UEF) is required for optimal transcription and is probably involved in Step I. Sarkosyl at 0.015 19) or at 0.05 in a crude extract inhibits Step II, the conversion of the pre-initiation complex to an initiation-competent complex. Higher concentrations of Sarkosyl inhibit Step III. and Roeder  31). Plasmid pML2GO was derived from pML(C2AT)19 by ligating into the SmaI site an AluI to SmaI restriction fragment of p C2AT)19, i.e. precisely the G-less region of the plasmid. The clone used, pML2GO, hadundergone a deletion and so contains a G-less region of approximately 620 bases; this is the length of the transcript obtained from SinaI-cut pML2GO or on ribonuclease Ti digestion of transcripts synthesized from uncut template DNA. In vitro Transcription Reactions: Transcription lysates were prepared from HeLa cellsas previously described  17), essentially by the method of Manley et al.  32). In an attempt to reduce theconcentration of endogenous nucleoside triphosphate the (NH4)2S04 precipitation step was repeated twice. The first (NH4)2S04 pellet was resuspended in a volume equal to that of the supematant from the high speed centrifugation and a second  NH4)2SO4 precipitation done by addition of 0.39 g/ml of solid (NH4)2S04. Transcription reactions were done in a final volume of 22.2 g1. Reactions contained either 13.3 g1 of transcriptionlysate  final concentrations of 12 mM 4- 2-hydroxyethyl)-1- 11109  Nucleic AcidsResearch piperazine ethane sulfonic acid (HEPES), pH 7.9; 7.5 mM MgCl2, 60 mM KCl, 0.2 mM EDTA, 1.2 mM dithiothreitol, 10 v/v) glycerol) or 7.5 gl of transcriptionlysate  final concentration 6.8 mM HEPES, pH 7.9;7.5 mM MgCl2, 34 mM KCl, 0.2 mM EDTA, 0.7 mM diothiothreitol, 5.7 v/v)glycerol). Reactionscontained 500 gm ATP, CTP and GTP, 50 ,um UTP, 4 pCi of [a-32P] UTP (BRESA, Australia) and 2 mM creatine phosphate except as indicated. When pML(C2AT)lg DNA was used as template GTP was generally omitted and 3-0-methyl GTP  Pharmacia) added to 200 jM  31). Histones and HMGs 1 and 2 were pre- incubatedwith DNAs  sequential 10 min incubations at 30°C, with histones being added prior to HMGs 1 and 2 if both were added) before addition of transcription lysate. Amounts of DNA template andchromosomal proteins added are indicated in individualfigurelegends. Sarkosyl NL97  Ciba-Geigy) was added to reactions as indicated. RNA was extracted from reaction mixtures and analysed by electrophoresis in 4 polyacrylamide gels containing 8 M urea  17). Transcription levels were quantitated by densitometryofautoradiographs exposed fordifferent times, with appropriate correction for 32p decayand for differences in recovery  estimated from internal DNA marker fragments). Ribonuclease U2 Digestions: Transcription reactions were stopped by addition of 50 p1 of stopbuffer, 25 mM Tris.HCl, pH 8, 25 mM EDTA, 1 SDS, 50 jg/ml Proteinase K followed by incubation for 2 hr at 37°C. 150 pl of 8 M urea in 10 mM Tris.HCl, pH 8, 1 mM EDTA containing a labeled DNA marker fragment was addedand the mix extracted with phenol:chloroform  1:1) and three timeswith chloroform prior to ethanol precipitation. The pellet was rinsed with 70 ethanol, dried and resuspended in 22 pl of digestionbuffer  22 mM sodium citrate, pH 3.5, 1 mM EDTA, 0.04 w/v) bromphenol blue, 0.25 w/v) Xylene cyanol FF,4.2 M urea containing 84 gg/ml yeast tRNA). 10 g1 was analysed on a 4 urea/polyacrylamide gel. To another 10 p1 was added2 pl  1 to 2 units) of ribonuclease U2  Pharmacia, sequencing grade) and the mix incubated for 2 to 3 hr at 55°C before loading directly on a 20 acrylamide, 7 M urea gel. Quantitation was as above. RESULTS We haveobserved that optimal stimulation of transcription in vitro by HMGs 1 and 2occurs when they are incubatedwith the template DNA prior to theaddition of transcriptionlysate  17). Therefore, DNA templates were pre-incubated with HMGs 1 and 2 at low ionic strength  10 mM Tris HCl, 1 mM EDTA, pH 7.9)prior to theaddition of transcription extract and adjustment to final buffer conditions. The DNA templates contained the adenovirus major late promoter sequences from -404 to +10 (pML(C2AT)19 and pML2GO) or from -61 to +192 (pAdMLP and pHIB); MLTF, which on binding tothe AdMLP protects the region from -65 to -50 from DNaseI, binds efficiently to both promoter fragments  F. Watt and P. Molloy, unpublished data)as bases essential for its binding lie 3 to base -62. 11110  Nucleic AcidsResearch 80 60 -   2o 0 0 0 0.1 0.2 0.3 0.40.50.6 HISTONES  ug) Figure 2. DNA Precipitation in the Presence of Histones, HMGs 1 and 2 and Transcription Lysate. Plasmid pAdMLP was linearized with BamHl and end-labeled. Samples of DNA  0.3 jg) were preincubated in 10 mM Tris HCI, 1 mM EDTA pH 7.9 successively for 10 min periods with the indicated amounts of histones then HMGs 1 and2 prior to addition of transcriptionlysate or buffer to bring the final conditions to those indicated in Materials and Methods when 13.3 X of lysate per 22 p1 reaction was used. After 30 min at 300 incubations were centrifuged at 11,000 x g for 2 min, aliquots of the supernatants taken and radioactivity determined following precipitation with trichloracetic acid and collection on glass-fibre discs.   a ) no HMGs 1 and 2,   ) 0.3 jg HMGs 1 and 2,   * ) 1 jig HMGs 1 and 2,   0 ) plus transcription lysate, no HMGs 1 and 2, ( O>) plus transcription lysate, 1 jg HMGs 1 and 2. Our previous data had also shown that HMGs 1 and 2 could stimulate transcription to a greater extent in reactions in which transcription hadbeen inhibited by histones  17). In these experiments histones were not specifically pre-formed into nucleosomes on template DNA, though some nucleosome formation may occur under in vitro transcription conditions  33,34,35). By thesequential addition of two template DNAs to reactions we have 11111
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