Footprinting of ribosomal RNA genes by transcription initiation factor and RNA polymerase I

Sep 2, 2026

Species-specific transcription initiation factor (TIF) and purified RNA polymerase I binding to the 39S ribosomal RNA gene promoter from Acanthamoeba were mapped using DNase I footprinting. Footprints correlated with TIF transcriptional activity and used labeled DNA as a template. TIF protects upstream promoter regions on coding and noncoding strands, while polymerase I extends protection only when TIF is present, indicating stable initiation complex formation. Adjacent protected regions support promoter recognition via both DNA-protein and protein-protein contacts.

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Abstract

Experimental Procedures

Preparation of TIF and RNA Polymerase I

DNA Templates

In Vitro Transcription

DNase I Footprinting

# Footprinting of ribosomal RNA genes by transcription initiationfactor and RNA polymerase I DNase I footprinting/ribosomal RNA gene promoter/in vitro transcription/Acanthamoeba castellani/eukaryotic RNA polymerase)ERIK BATEMAN,CALVIN T.IIDA,PREECHA KowNIN,AND MARVIN R.PAULE* Department of Biochemistry and the Cellular and Molecular Biology Interdisciplinary Program,Colorado State University,Fort Collins,CO 80523Communicated by Marshall Fixman,August 12,1985 ABSTRACT The binding of a species-specific transcrip-tion initiation factor(TIF)and purified RNA polymerase I tothe promoter region of the 39S ribosomal RNA gene fromAcanthamoeba were studied by using DNase I“footprinting.”Conditions were chosen such that the footprints obtained couldbe correlated with the transcriptional activity of the TIF-containing fractions used and that the labeled DNA presentwould itself serve as a template for transcription.The tran-scription factor binds upstream from the transcription startsite,protecting a region extending from around-14 to-67 onthe coding strand,and-12 to -69 on the noncoding strand.The protein that binds to DNA within this region can becompeted out by using wild-type promoters but not by usingmutants which do not stably bind the factor.RNA polymeraseI can form a stable complex in the presence of DNA andtranscription factor,allowing footprinting of the completetranscription initiation complex.RNA polymerase Iextends theprotected region obtained with TIF alone to around+18 on thecoding strand,and to+20 on the noncoding strand.This regionis not protected by polymerase Iin the absence of TIF.The closeapposition of the regions protected by TIF and polymeraseprovides evidence that accurate transcription of the ribosomalgene may be achieved through protein-protein contacts as wellas through DNA-protein interactions. Transcription initiation of eukaryotic genes in vitro requiresthe presence of at least one protein factor in addition to RNApolymerase and a promoter-containing DNA fragment(1-3).For class II,III,and possibly class I genes,one or more ofthe transcription factors acts through stable interaction withthe gene promoter regions(4-6),allowing correct initiationby the polymerase.The details of this process differ consid-erably between different gene classes,with respect to thesequence and positioning of promoter regions as well as thenumber of factors thought to be required for transcription. Control regions for polymerase II,and to some extentpolymerase III,show promoter sequence homology whencomparisons between species are made(7,8).In contrast,thepromoter sequences involved in polymerase I transcriptionare highly diverged,making identification of regulatorysequences by comparison of conserved regions difficult.Studies on ribosomal gene promoters do,however,show thata region flanking the 5'side of the initiation start site isrequired for transcription(9).Part of this region functions byinteraction with protein components of the system to form astable complex that commits the template for correct tran-scription(6,10-12).In the Acanthamoeba rRNA genes,thesequence requred for template commitment extends fromaround-20 to-47,and it can be divided into two regions:one(A region)is absolutely required for transcription,andthe other(B region)is involved with the stability of a preinitiation complex formed between the DNA and a tran-scription initiation factor(TIF)(6).A third region flanking thestart site is important for initiation by polymerase I(6).Sincepolymerase is unable to initiate transcription in the absenceof transcription factor(s),it is likely that at least part of thecontrol region functions through binding of factors,with adistinct region serving as a polymerase binding site. DNase I'"footprinting”(13)has been used as a probe fora wide variety of DNA-binding proteins,allowing identifica-tion of those DNA sequences that are in a stable complex.Wehave utilized this approach to determine the DNA bindingsites both

FAQs

How does RNA polymerase I alter the footprint compared with TIF alone?

With TIF present, RNA polymerase I extends the protected region to about +18 on the coding strand and +20 on the noncoding strand; polymerase I does not protect that region in the absence of TIF.

Sep 2, 2026

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