Fractionation of transcription factors for RNA polymerase II from Drosophila Kc cell nuclear extracts.

D.H Price,A.E Sluder,A.L Greenleaf

doi:10.1016/s0021-9258(18)61497-8

Abstract

Drosophila Kc cells were utilized to prepare nuclear extracts in which promoter-containing DNA templates were efficiently transcribed by RNA polymerase II. A combination of fractionation schemes was used to identify and partially purify seven activities (factors) which affected the transcription of four different genes in vitro. Reconstructing specific transcription required exogenous RNA polymerase II in addition to these factors. Moreover, the high efficiency of transcription characteristic of the crude extract was preserved in reconstruction reactions. The methods used are presented in detail. Functions were assigned to several of the factors. One essential factor appeared to affect initiation and displayed chromatographic properties unlike any other Drosophila transcription factor previously described. Two factors specifically affected RNA chain elongation. Another activity was a DNase inhibitor required to preserve template integrity in the fractionated system. The remaining three factors were not absolutely essential but affected the specific in vitro transcription either qualitatively or quantitatively. A comparison of these transcription factors with other Drosophila and mammalian transcription factors is made.

Highlights

IntroductionTwo factors affected RNA chain polymeraseI1 becomesassociated, forming satable initiation elongation
0.1 L 4 . 6 ographicfractions arerecombined with purified RNA polymeraseI1 to reconstrucet fficient specific transcription
Methodologyof TranscriptionFactor Fractionation-A complication encountered in fractionating crude teoxtorabcttasin transcription factors is the presenocfeactivities thataffect FIG. 12

Summary

Introduction

Two factors affected RNA chain polymeraseI1 becomesassociated, forming satable initiation elongation. Another activitywas a DNase inhibitor complex(Fireet al., 1984;SawadogoandRoeder,1985a;Safer required to preservetemplateintegrity in the fraction- et al.,1985).This step is onwehich requiresthe hydrolysisof ated system. Crudeextractsof cells contain many activities that affect transcription nonspecificallyand, seriously complicate purificationof specificfactors.The crude extracts from HeLa cells that werethe starting materiafolr the early fractionationwork (Matsuiet al., 1980;Samuelset al., 1982) were very inefficient at transcribingexogeneoustemplates. This wasdueto thepresenceof inhibitors of transcription as well asto the low concentrationof specific factors. In order tboe able toinvestigatethe functional consequencesof mutationsin genes for Drosophila RNA polymeraseI1 subunits(Greenleafet al., 1980;Mortin and Lefevre, 1981;Voelker et al, 1985,for example)w, e soughtto prepareRNA polymeras1e1-dependenttranscription extracts

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Conclusion