Proc. Nati. Acad. Sci. USAVol. 80, pp. 4238-4242, July 1983Biochemistry

Topography of transcription: Path of the leading end of nascentRNA through the Escherichia coli transcription complex

(photoaffinity labeling/RNA polymerase subunits/bacteriophage T7 Dill and D123 DNA/Al promoter/cleavable reagent)

MICHELLE M. HANNA* AND CLAUDE F. MEARESt

Department of Chemistry, University of California, Davis, California 95616

Communicated by John D. Baldeschwieler, April 4, 1983

ABSTRACT A cleavable dinucleotide photoaffinity reagent wasprepared and used to map the path of the leading end of the RNAtranscript across the surface of Escherichia coli RNA polymerase/T7 DNA transcription complexes. By using 5'-(4-azidophenacyl-thio)phosphoryladenylyl(3'-5')uridine, transcription was specifi-cally initiated at the Al promoter of bacteriophage T7 DIll orD123 DNA. Transcription complexes containing radiolabeled RNAchains of various lengths (4-116 nucleotides) were prepared, andthe 5' end of the RNA transcript was then covalently attached tothe nearby polymerase subunits or DNA by irradiation with UVlight. The photoaffinity-labeled enzyme subunits and DNA wereseparated, the radiolabeled RNAs were cleaved from each, andthe lengths and sequences of RNA attached to each componentwere determined. The leading end of RNA chains up to 12 baseslong was found to label the DNA and the ,3 and /3' subunits ofRNA polymerase, with more than 90% of the label going to theDNA. When the RNA transcript reached 12 bases in length, the5' end diverged from the DNA and only the /8 and /3' enzyme sub-units were labeled thereafter. These two subunits were heavilylabeled by RNA chains 12 to as many as 94 bases long. No sig-nifitant labeling of the a subunit occurred. The a subunit was notlabeled by RNAs longer than the trinucleotide.

DNA-dependent RNA polymerases play a key role in geneexpression. Escherichia coli RNA polymerase, the most exten-sively studied, contains five major subunits and has a Mr of ap-proximately 454,000 (1). The "core" enzyme consists of sub-units A3' (Mr, 160,000), /3 (Mr, 151,000), and two a subunits (Mr,36,000) and is capable of RNA elongation after transcription hasbeen initiated. The holoenzyme contains the core plus the dis-sociable subunit or (Mr, 70,000) which is required for specificrecognition of promoter sites on DNA. Various approaches havebeen taken to investigate the molecular topography of the en-zyme as well as the changes that occur upon binding of DNAand initiation of transcription. Chemical modification studieshave provided information about the contact points betweenpolymerase subunits (2, 3), between the polymerase and DNA(4-8), and between the polymerase and substrate analogs ornascent RNA (9-16). The susceptibility of the polymerase sub-units to attack by proteases (17-19) has been investigated in boththe isolated enzyme and in binary complexes with DNA. In ad-dition to these chemical studies, physical methods such as small-angle x-ray scattering (20-23) and neutron scattering (24, 25)have provided information on the size and shape of the en-zyme.

Little is known about the path of a growing RNA chain throughthe transcription complex that causes its synthesis. Here wedescribe experiments by which the contacts of the leading endof the nascent RNA with RNA polymerase subunits and DNA

The publication costs of this article were defrayed in part by page chargepayment. This article must therefore be hereby marked "advertise-ment" in accordance with 18 U.S.C. § 1734 solely to indicate this fact.

have been determined. Our method involves the use of a cleav-able photoaffinity probe, containing both a photosensitive azidemoiety for covalent attachment and a selectively cleavable S-Pbond for subsequent removal of the attached oligonucleotide.We chose to prepare the dinucleotide photoaffinity reagent weused (Fig. 1) for three reasons: dinucleotides initiate transcrip-tion much more efficiently than mononucleotides (26); the ar-omatic azide moiety produces a chemically reactive, relativelynonselective nitrene (27) upon illumination with wavelengthsgreater than 300 nm; and the S-P bond was found to be stableunder various conditions, although it could be cleanly hydro-lyzed in the presence of organomercurials.

At the initiation of transcription, the photoaffinity probe isincorporated into the leading (5') end of a nascent RNA chain.Because addition of nucleoside triphosphates occurs at the 3'end of the growing RNA chain in the elongation site of the en-zyme, the 5' end must move out over the surface of the en-zyme-DNA complex as transcription proceeds. Transcriptioncomplexes containing RNA chains of almost every length from4 to 116 nucleotides have been prepared by using RNA chainterminators that substitute for ATP, CTP, GTP, or UTP to ter-minate transcription specifically at different positions. Photo-lysis of the transcription complexes followed by separation ofcomponents and analysis of attached RNA chains provided amap of the transcript's path through the complex (Fig. 2).

MATERIALS AND METHODSMaterials. E. coli MRE 600 cells were purchased from Grain

Processing, Muscatine, Iowa. RNA polymerase was purified bythe method of Burgess and Jendrisak (28) as modified by Loweet al (29). p-Azidophenacyl bromide was purchased from Pierceand 3'-O-methylnucleotides were from P-L Biochemicals. Affi-Gel blue resin was purchased from Bio-Rad. Adenosine 5'-O-thiomonophosphate was purchased from Boehringer Mann-heim. 3'-Deoxyadenosine triphosphate (cordycepin triphos-phate) and other nucleotides were from Sigma. Radiolabelednucleotides were purchased from Amersham.

Bacteriophage T7 D111 and D123 DNAs were kindly pro-vided by Judy Levin and Michael Chamberlin. Each of theseDNAs has only one strong promoter site (Al) for E. coli RNApolymerase, and the RNA transcripts have identical sequencesfor the first 23 nucleotides. The sequence of the D123 tran-script is 5'-A-U-C-G-A-G-A-G-G-G-A-C-A-C-G-G-C-G-A-A-U-A-G-U-G-A-G-A-A-C-U-U-G-G-C-G-A-G-A-G-A-A-C-A-A-C-C-U-C-G-A-A-C-G-C-C-G-C-A-A- G-G-A-C-A-A- G-A-G-A- G-G-G-C-G-G-C-G-U-G-G-C-A-U-A-G-A-C-G-A-A-A-G-G-A-A-

Abbreviation: N3RSpApU, 5'-(4-azidophenacvlthio)phosphorylade-nylyl(3'-5')uridine.* Present address: Dept. of Biochemistry, Univ. of California, Berke-ley, CA 94720.tTo whom reprint requests should be addressed.

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Proc. Natl. Acad. Sci. USA 80 (1983) 4239

IH20 QI d

N391-C -CH2-S-P-O-CH2< 2

)HgOAc 0 OH

eOP=O

OCH2

OH OH

FIG. 1. Cleavabledinucleotidephotoaffinityprobe5'-(4-azidophen-acylthio)phosphoryladenylyl(3'-5')uridine [N3RSpApU], showing theS-P bond that is hydrolyzed in the presence of phenylmercuric ace-tate.

A-A-G-G-U-U-A-A-A-G-C-C-A-A-G-A-A-A-C-U-C-G-C-C ....Buffers. Buffer A: 0.25 M Tris HCI, pH 7.9/0.025 M 2-mer-

captoethanol/0.05 M NaCI/0.05 M MgCl2/25% (vol/vol) glye-erol/5 mM K2HPO4. Buffer B: 8 M urea/50% (wt/vol) su-crose/1.2% (wt/vol) NaDodSO4/0.15% bromphenol blue/70mM triethanolamine/50 mM HCI, pH 7.5. Buffer C: 0.44%ethanolamine/0.45% glycine/0.1% NaDodSO4, pH 9.7.

Synthesis of 5'-(4-azidophenacylthio)phosphoryladenylyl-(3'-5')Uridine (N3RSpApU). The dinucleotide photoaffinityprobe (Fig. 1) was prepared by alkylation of 5' (thiophospho-ryl)adenylyl(3'-5')uridine (SpApU) with azidophenacyl bro-mide (30). The product was incubated with phenylmercuricacetate at neutral pH to stimulate hydrolysis of the S-P bond,and the time for complete hydrolysis was determined.

Ternary Transcription Complex Preparation (RNA C 20Bases). All transcription reactions were carried out in dim light.Transcription complexes containing radiolabeled RNA chains of

4-20 nucleotides were prepared in three separate reactions, eachcontaining a different RNA chain terminator (3'-O-methyl-CTPor 3'-O-methyl-GTP or 3'-dATP). None of the reaction mix-tures contained UTP, which is required at transcript position21. All reaction mixtures contained the following in 125 ,Al: 8nM DNA, 16 nM RNA polymerase, 12.5 jul of buffer A, 100MM N3RSpApU, 5 jxM [a-32P]GTP (200 Ci/mmol; 1 Ci = 3.7x 1010 Bq), 5 ,iM [a-32P]CTP (200 Ci/mmol), 1 MM ATP, andone terminator (3'-deoxy-ATP at 0.1 mM, 3'-O-methyl-CTP at0.5 mM, or 3'-O-methyl-GTP at 0.25 mM). The buffer, DNA,RNA polymerase, and N3RSpApU were preincubated at 370Cfor 5 min before addition of nucleotides. After addition of nu-cleotides, transcription was allowed to proceed at 370C for 5min prior to photolysis.

"Total RNA" Samples. Samples were withdrawn from eachreaction mixture prior to photolysis for analysis on polyacryl-amide gels. Aliquots (10 t'l) were added to 20 Ml of saturatedurea and 20 Ml of saturated phenylmercuric acetate in 0.1%NaDodSO4. These were allowed to hydrolyze to oligonucleo-tide 5'-monophosphates overnight in the dark at room tem-perature. Then 5 Ml of 1 M dithiothreitol was added to reduceazide to amine and, after 1 hr at room temperature in the dark,the samples were adjusted to 0.05% bromphenol blue and 0.05%xylene cyanol before electrophoresis.

Photolysis and Component Separation. Transcription mix-tures were transferred to 6 x 20 mm borosilicate glass tubesand irradiated for 2 min in a Rayonet type RS photochemicalreactor (A> 300 nm). The reaction mixtures containing RNAs<20 bases long were treated with 20 Ml of buffer B and 10 Mlof 1 M dithiothreitol, and allowed to sit for 1 hr at room tem-perature in the dark to reduce any remaining azide. The sam-ples then were loaded onto 40 cm X 0.75 mm NaDodSO4/urea(31) step gradient gels in buffer C (32 cm of 3.5% acrylamideas upper layer; 6 cm of 7% acrylamide as lower layer). Elec-trophoresis was carried out for 8 hr at 30 mA. This was suffi-cient to achieve separation of the enzyme subunits and the DNA

-N)

DNA

>o> xMx-x

ieI3 CT

X()2-n O XX(X) _nOn XX

41 N1XX(X)n

U

41 41

a

0

2=

:::::

XX(X)o -

xxxxxxxxx-xxx -

FIG. 2. Experimental strategy for determining the path of the leading (5') end ofRNA through the transcription complex. First, transcriptioncomplexes containing RNAs of various lengths are formed. The drawing shows chain lengths in which (i) the 5' end ofRNA is in contact with boththe enzyme and the DNA or (ii) the 5' end has diverged from the DNA but still contacts the enzyme or (iii) the 5' end of the RNA has left the surfaceofthe enzyme. After irradiation ofthe transcription complex to attach theRNA covalently to surrounding protein orDNA, the complex is dissociatedand the components are separated by gel electrophoresis. The radiolabeled RNA chains are cleaved from the DNA and protein subunits in the pres-ence of phenylmercuric acetate and the chain lengths of the released RNAs are determined by a second gel electrophoresis (X, a ribonucleotideresidue). In practice, four reactions are done in parallel; each mixture contains a base-specific terminator (A, C, G, or U), so that the base sequenceof the transcript is verified.

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4240 Biochemistry: Hanna and Meares

A C G

DNA A0.

(3 ;5

CT

FIG. 3. Autoradiogram showing separation of the components ofthe transcription complex by polyacrylamide gel electrophoresis afterphotoaffinity labeling with radioactive RNA chains containing 4-20bases. Bands visualized by staining are identified in the left margin.The three lanes contained terminators that substituted for ATP, CTP,and GTP (see Fig. 4). Note that ,B and ,B' are reasonably well resolvedand that ,B' moves farther down than ,B in this NaDodSO4/urea system(30).

(Fig. 3). To eliminate the possibility of nonspecific labeling,appropriate control reactions, each lacking one component ofthe transcription reaction, were also run.

For the reaction mixtures containing RNAs >20 bases longit was necessary to separate the ,B and A' enzyme subunits be-

fore electrophoresis because the attachment of long RNA mol-ecules to these subunits causes band spreading and overlap.Separation was accomplished by binding the f3' subunit to Affi-Gel blue resin in 7 M urea with a method similar to that of Wuet aL (32). Electrophoresis was then carried out as describedabove. Because long RNA chains comigrate with the a subunit,the a band was excised from the gel, placed at the top of anRNA sequencing gel (see below), and separated from contam-inating RNAs by electrophoresis. The a subunit was retainedat the top of the RNA sequencing gel.

Electroelution and Cleavage Reactions. Radiolabeled DNAand RNA polymerase subunits were located by autoradiographyof the gels and comparison to stained marker holoenzyme. Theautoradiogram was laid over the gel and the radiolabeled com-ponents were excised. The gel pieces were placed in dialysisbags with 0.2 ml of 0.1% NaDodSO4 and electroeluted for 3 hrat 50 mA. The solutions containing electroeluted macromole-cules were then removed, and 0.2 ml of saturated phenyl-mercuric acetate in 0.1% NaDodSO4 containing carrier RNA (1mg/ml) was added to each. The samples were allowed to hy-drolyze (in order to liberate oligonucleotide 5'-phosphates) bystanding at room temperature for 24 hr. The samples were ly-ophilized and resuspended in 40 tkd of 7 M urea, marker dyeswere added, and 20-Ad aliquots. were loaded onto RNA se-quencing gels.RNA Sequencing Gels. Total RNA samples and RNA cleaved

from protein subunits and DNA were analyzed on 40 cm X 0. 75mm 25% acrylamide gels [0.089 M Tris borate, pH 8.3/2.5 mMEDTA/7 M urea/1:29 methylenebis(acrylamide)]. Gels wereallowed to polymerize for 12 hr and then were subjected to 1,000V for 6 hr before use. Electrophoresis was at 1,000 V until thebromphenol blue marker had migrated 27.5 cm from the bot-tom of the sample well (Fig. 4) or until the xylene cyanol hadmigrated to the bottom of the gel (Fig. 5). Autoradiography ofgels was performed at -79°C with Kodak X-Omat AR-5 x-rayfilm and Cronex Lightning Plus intensifying screens.

GG A C G

TotalRNA

FIG. 4. RNA sequencing gelsshowing the RNA chains contain-ing 4-20 bases which were re-leased from the components of thetranscription complexes separatedin Fig. 3. The lanes labeled A, C, orG contained RNA chains termi-nated with 3'-deoxy-ATP, 3'-O-methyl-CTP, or 3'-O-methyl-GTP,respectively. The "Total RNA' lane

* 20 A contained equal aliquots from eachU 19 A of these reactions (before photol-

17 C ysis) and shows the distribution ofRNA chains present in transcrip-

14 c tioncomplexes (or as released abor-12 C tive initiation products). The num-11 A bers identify the RNA chain length9 G of each band, and the letters iden-8 G tify the base at which the chain

terminated. The radioactivity at thej 7 A top of the DNA panel is due to the6 G DNA itself, which becomes radio-

, 5 A labeled in the presence of RNApolymerase and substrates (11, 30).

4 G The bands near 20A in the a panelare artifacts due to comigration offree RNA with the a subunit.

Ur

A CDNA

.04u

012tl

A C G

20 -

- 19

17

3143 12

A C G

20O1,9 * 17

* 14

4.9

7

.* 5 .

4

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Proc. Natl. Acad. Sci. USA 80 (1983) 4241

DNA BETA PRIMEA C G U A C G u

94

8F

BETAA C G U

.. ..6

SIGMA ALPHAA C 0 1J A C G U -

2

-7 i.

* ;-4:

I -G

S -U

' ;.;S FIG. 5. RNA sequencing-ok gels showing the 5' contacts of

RNA chains containing 21-116* 6^ bases in E. coli RNA polymer-

ase/T7 D123 DNA transcrip-* A tion complexes (see Fig. 4).%; - - f

RESULTSContacts by RNA Chains of 4-20 Nucleotides. The DNA

and RNA polymerase contacts made by the 5' ends of RNA chainscontaining 4-20 bases are shown in Fig. 4. The major RNA spe-cies represented are lengths 5, 7, 11, 19, and 20 (3'-deoxy-ATPterminated), 12, 14, and 17 (3'-O-methyl-CTP terminated), and4, 6, 8, and 9 (3'-O-methyl-GTP terminated). Unterminated20-mer can occur in all three lanes because the next substrate,UTP, was absent. The production of RNA of a given length de-pends on several factors, including "pausing" of the enzyme atcertain sites on the template, overproduction of short oligo-nucleotides due to "abortive initiation," and the concentrationand efficiency of each terminator (33). RNAs present in abun-dance led to detectable photoaffinity labeling (up to the 94-mer),but the labeling yield varied; a distinct increase in overall la-beling yield occurred for RNAs longer than 11 nucleotides.

The distribution of label among the DNA and polymerasesubunits was determined by densitometer scanning of auto-radiograms or by excision and scintillation counting of the gelpieces. More than 90% of the photoaffinity labeling by chainlengths 4-11 occurred on the DNA, and the remaining labelwas distributed between the /3 and A3' subunits. Upon elon-gation of the RNA chain to the 12-mer, the photoaffinity labeldistribution shifted such that the DNA labeling decreased to< 10% with a corresponding increase in /3 and A3' labeling. Fur-ther elongation of the RNA to the 14-mer caused a shift of vir-tually all (>99%) of the photoaffinity label to the /3 and A3' sub-units. RNA chains 14, 17, 19, or 20 nucleotides long labeledonly these two subunits. As shown in Fig. 4, the leading endof the RNA makes no significant contacts with the or or a sub-units.

Contacts by RNA Chains Longer Than 20 Nucleotides. TheRNA sequencing gels identifying DNA and enzyme contactsmade by RNA chains of more than 20 nucleotides on T7 D123DNA are shown in Fig. 5. The 5' ends of all RNA chains longerthan 20 nucleotides labeled both the /3 and /3' subunits; the /3'subunit consistently was more heavily labeled than the /3. The94-mer was the longest RNA to give significant labeling, eventhough RNAs as long as 116 nucleotides were present; thisprobably indicates that the 5' end of the RNA has left the sur-face of the enzyme and reacted entirely with the solvent (27).No significant (>1%) photoaffinity labeling of the DNA or thea or a subunit occurred.

DISCUSSIONIn addition to the experiments in Figs. 4 and 5, we photoaf-finity-labeled this transcription complex with a trinucleotide (30);the DNA received roughly 90% of the label and the /3 and oCsubunits also were labeled. In an earlier study using a similarazide dinucleotide probe and a poly[d(A-T)] template, the 5'end of the trinucleotide was found to contact the /3' and af sub-units of the enzyme (11). However, subsequent work has shownthat the /3 and 3' subunits invert in the NaDodSO4/urea sys-tem (31) used in ref. 11, relative to their mobilities in standardLaemmli gels (34). Thus, it is /3 rather than /' that is labeledby the trinucleotide (30).

In the earlier work (11), the tetranucleotide was found to la-bel both the /3 and /3' subunits and not to label the o subunitsignificantly. These are the same enzyme contacts we now seeon the T7 Al promoter. It appears that the 5' ends of only thedinucleotide (11) and trinucleotide (11, 30) contact the or sub-unit significantly. The RNA chain length at which af is releasedfrom E. coli RNA polymerase has been estimated to be ninebases (35).

The results of the experiment in Fig. 4 are in good agree-ment with other work concerning DNA strand separation intranscription complexes. The number of DNA base pairs un-wound in transcription complexes on T7 DNA has been re-ported to be 10-17 (36-39). The length of the DNA region thatis melted appears to be at least 12 base pairs because our resultssuggest that the 5' end of the transcript is involved in a DNARNAhybrid until the RNA chain exceeds 12 bases in length. Theabrupt separation of the transcript from the DNA at a length

DNA|I

/3'

a

0

+

5 10 15 20 80 8590 95100RNA CHAIN LENGTH

FIG. 6. Contacts of the leading (5') end ofRNA in E. coliRNA poly-merase/T7 D123 DNA transcription complexes. Thick lines showheaviest labeling.

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4242 Biochemistry: Hanna and Meares

of 12-14 nucleotides is evident in Fig. 4. Protection of a 12-base RNA fragment from ribonuclease has been reported (40).

Almost the entire path of the nascent RNA chain lies alongthe interface between the /3 and A3' subunits. Detectable la-beling of both subunits starts at the tetramer, although the la-beling of these subunits by RNA chains of 4-11 nucleotides issmall compared to the labeling of the DNA. It appears (Fig. 5)that the labeling of both subunits continues until the leadingend of the RNA chain leaves the surface of the transcriptioncomplex.No significant (> 1%) photoaffinity labeling of the a subunits

has been found to occur. Because some of the free oligonu-cleotides comigrate with a during electrophoresis in low-per-centage NaDodSO4 gels, study of the a subunit is particularlydifficult. We used a second electrophoresis in 25% polyacryl-amide to improve the separation of a from oligonucleotides.The results of these experiments are summarized in Fig. 6.

This provides a complete description of the transcription com-ponents contacted by RNA during its synthesis on a T7 D123template (Al promoter site). To ensure the reliability of thisinformation, control experiments were included at every step,and all experiments were designed to avoid ambiguity. In ad-dition to the precautions described in refs. 11 and 30, the useof a cleavable probe, a DNA template with a single promotersite, and four base-specific terminators allowed us to examineboth the length and the base sequence of RNA that had beencovalently attached to transcription components by photoaffin-ity labeling.

The literature contains some brief communications of ex-periments to determine the beginning of the path of nascentRNA on RNA polymerase (13-15). When they addressed thesame points, those results did not always agree with ours. In theonly report in which an effort was made to identify the oh-gonucleotides responsible for labeling (13), transcription wasinitiated from multiple promoter sites on T7 DNA. Only onereaction (unterminated) was used, involving a P-N bondedprobe which was cleaved off in 10% formic acid. The pho-toaffinity labeling of DNA was not investigated, and only RNAsshorter than 13 nucleotides were produced. The authors did notobserve any labeling of the P3' subunit [although this had beenreported previously (14, 15)] and reported the labeling of only3 by RNAs 2, 6, and 12 nucleotides long and o by RNAs 2 and6 nucleotides long (13). As shown in Fig. 6, our results are sig-nificantly different from this. It may be that different DNApromoter sites lead to slightly different paths for the transcriptor, possibly, that different photoaffinity probes give somewhatdifferent results.

It is also possible that enzyme and DNA contacts will dependon the base sequence of the transcript (e.g., because of RNAsecondary structure). As a preliminary test of this, we havecompared two mutant T7 DNAs (D111 and D123). These mu-tants both contain the Al promoter and have the same tran-scribed base sequence until residue 23, after which the tran-scribed sequences are quite different. For RNA transcripts 3-20 nucleotides long, the two DNA templates lead to identicallabeling results (Fig. 4; ref. 30). For RNAs longer than 20 nu-cleotides, D111 DNA gives results similar to those with D123(Fig. 5), except that there are some indications that D111 tran-scripts 45-50 nucleotides long again label the DNA. Furtherstudies are needed to confirm this result.

We thank Judy Levin, Michael Chamberlin, Susan Bernhard, LeslieDeRiemer, Michael McCall, and Lyle Rice for gifts of material, helpfuldiscussions, and technical assistance. We thank Roy. Doi, Joel Keizer,and Carl Schmid for critically reading the manuscript. This work was

supported by Research Grant GM25909 and Research Career Devel-opment Award CA00462 to C.F.M. and, in part, by Training GrantGM07377-05 to M.M.H. from the National Institutes of Health.

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by g

uest

on

Apr

il 30

, 202

0