Transcription Termination by Bacteriophage T7 RNA Polymerase at ...

Transcription Termination by Bacteriophage T7 RNA Polymerase at ...


Transcription termination by

bacteriophage T7 RNA polymerase at

rho-independent terminators.

S T Jeng, J F Gardner and R I Gumport

J. Biol. Chem. 1990, 265:3823-3830.

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0 1990 by The American Society for Biochemistry and Molecular Biology, Inc.

Vol. 265, No. 7, Issue of March 5, pp. 3823-3830. 1990

Printed m U.S. A.

Transcription Termination by Bacteriophage T7 RNA Polymerase at

Rho-independent Terminators*

Shih-Tong Jeng& Jeffrey F. Gardnerj, and Richard I. GumportSll

(Received for publication, August 30, 1989)

From the SDepartment of Biochemistry, College of Medicine and School of Chemical Sciences and the $Department of

Microbiology, School of Life Sciences, University of Illinois, Urbana. Illinois 61801

We have investigated the mechanism of transcription

termination by T7 RNA polymerase using templates

encoding variants of the transcription-termination

structure (attenuator) of the regulatory region of the

threonine (thr) operon of Escherichia coli. The thr

attenuator comprises the following two distinct struc-

tural elements: a G+C-rich inverted repeat, which en-

codes au RNA hairpin structure, and A+T-rich regions,

one of which contains a continuous sequence of tem-

plate deoxyadenosine residues within which the tran-

script terminates. Fourteen attenuator variants were

analyzed and we find that not only the hairpin struc-

ture itself but also its sequence influences termination.

Furthermore, the formation of a hairpin in the RNA

encoded by the A+T-rich regions of the attenuator is

not mandatory for termination.

A series of seven deletion variants that successively

shorten the deoxyadenosine tract in the attenuator

template were also analyzed. Results from these exper-

iments indicate that complete readthrough occurs

when there are four or fewer deoxyadenosine residues.

With 5 template deoxyadenosine residues there is 5%

termination increasing to 32% with 8 deoxyadeno-

sines, the value produced by the wild-type attenuator.

In addition, a comparison with E. coli RNA polymerase

shows that T7 RNA polymerase requires a more per-

fect region of dyad symmetry and a longer deoxyaden-

osine tract than does the bacterial enzyme to terminate

with maximum efficiency.

The RNA polymerase encoded by bacteriophage T7 is a

single polypeptide that recognizes a highly conserved promo-

tor (l-3). T7 RNA polymerase initiates and elongates tran-

scripts more efficiently than Escherichia coli RNA polymerase

(1, 4, 5). The enzyme produces full length transcripts from

DNA templates containing a T7 promotor (6), and vectors for

the high level expression of genes (6, 7) and of other DNA

sequences (8) cloned behind T7 promoters have been devel-


Some DNA templates contain sequences that lead to the

termination of transcription by T7 RNA polymerase. The

rrd terminator and thr attenuator are rho-independent ter-

minators that stop both E. coli RNA polymerase (9-16) and

T7 RNA polymerase efficiently (this work). In addition, the

* This work was supported by National Institutes of Health grant

GM28717 (to J. G. and R. G.). The costs of publication of this article

were defrayed in part by the payment of page charges. This article

must therefore be hereby marked “advertisement” in accordance with

18 U.S.C. Section 1734 solely to indicate this fact.

VTo whom correspondence should be addressed: Dept. of Biochem-

istry, 415 Roger Adams Laboratory, University of Illinois, 1209 W.

California St., Urbana, IL 61801.


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E. coli rrnBT1 terminator, another rho-independent termi-

nator, also terminates T7 RNA polymerase in vitro (17, 18).

The T7 late terminator, T& located between genes 10 and 11

on the T7 genome, is structurally similar to the rho-inde-

pendent terminators of E. coli in that it contains a G+C-rich

region of dyad symmetry followed by a template deoxyaden-

osine tract (3), but it inefficiently terminates T7 RNA polym-

erase (19) either in vitro or in uiuo (20, 21). T7 RNA polym-

erase fails to terminate at Te, a rho-independent terminator

for the bacterial RNA polymerase that is also present in the

T7 genome (19-22). Thus, not all transcription termination

structures with the rho-independent structural motif effec-

tively terminate the phage polymerase. When the calculated

free energies for melting of these rho-independent structures

are compared, there is no direct correlation between termi-

nation efficiency of T7 RNA polymerase and the RNA helix


In addition to the familiar rho-independent terminators,

Mead et al. (7) reported that a template sequence containing

a discontinuous run of deoxyadenosine residues without an

apparent G+C-rich dyad-symmetrical structure causes 90%

termination with T7 RNA polymerase. When four of these

deoxyadenosines were changed to GCGC by site-directed mu-

tagenesis, the termination efficiency of T7 RNA polymerase

decreased to 10%. Termination at sites on DNA that lack the

known features of transcription-termination structures indi-

cates our incomplete understanding of the mechanisms spec-

ifying the termination event. With the aim of better under-

standing the features of rho-independent transcription ter-

mination structures that might influence termination of T7

RNA polymerase, we have studied the enzyme with the well

known attenuator structure from the regulatory region of the

threonine operon of E. coli.

Termination at the thr attenuator has been studied in detail

with E. coli RNA polymerase (11-16). The thr attenuator is

structurally similar to other rho-independent terminators (12,

16), and 90% of E. coli RNA polymerase molecules terminate

in vitro at this site (12). Several variants in the thr attenuator

have been constructed or selected by genetic procedures. They

include point substitutions in the G+C-rich region of dyad

symmetry (12, 13, 16), point substitutions in the A+T-rich

region (X5), and a set of nested deletions in the template

deoxyadenosine tract (12). It was convenient for us to use

these variants plus others to &udy transcription termination

with T7 RNA polymerase.

In this paper, we report the effects of 14 point variants in

the G+C-rich and A+T-rich regions and seven deletions in

the deoxyadenosine tract of the thr attenuator on transcrip-

tion termination of T7 RNA polymerase. The results indicate

that the sequence within the G+C-rich region, the stability

of the encoded RNA hairpin, and the length of the template

deoxyadenosine tract each affect the termination efficiencies.

3824 Rho-independent Termination of T7 RNA Polymerase

In contrast with E. coli RNA polymerase and under conditions

optimized for RNA symthesis, T7 RNA polymerase requires

a more stable RNA hairpin structure and a longer template

deoxyadenosine tract to terminate with maximum efficiency.


Materials-The labeled ribonucleoside triphosphate, [w~‘P]CTP

(410 Ci/mmol) was purchased from Amersham Corp. Unlabeled ri-

bonucleoside triphosphates were obtained from Sigma. T7 RNA

polymerase, restriction enzymes, and T4 DNA ligase were purchased

from Bethesda Research Laboratories. A Sequenase” kit was pur-

chased from United States Biochemicals, and the large proteolytic

fragment of E. coli DNA polymerase I was from Boehringer Mann-

heim GmbH.

Plasmids, Bacterial Strains, and Plasmid Purification-The plas-

mids used in this study are listed in Table I. The plasmids pTZ-I9u

and pTZ-18~ were gifts from B. Kemper (University of Illinois). All

plasmids were grown in E. coli HBlOl cells, and purified by successive

CsCl-ethidium bromide isopycnic centrifugations (23). After lineari-

zation with the appropriate restriction endonuclease, the DNA was

extracted with phenol and with chloroform/isoamyl alcohol 24:l.

After ethanol precipitation and centrifugation, the DNA pellets were

dried under vacuum and dissolved in water for use in transcription


Construction of Templates-The flow charts for the construction

of the DNA templates used in this study are shown in Fig. 1, A and

B. A plasmid (pTZ-19tt) containing the T7 promotor, thr attenuator

and rrnC terminator was constructed as follows (Fig. L4). A BamHI-

MluI fragment (43 base pairs) from bacteriophage lambda (base pairs

5505 to 5548 (24)) was inserted into the BamHI and MluI sites of

bacteriophage M13mp9 (3-6) replicative-form DNA (11) to generate

M13mp9:X43 encoding the transcription-termination structure but

not the antiterminator of the thr operon regulatory region (11, 12,

16). A TuqI fragment (103 base pairs) from M13mp9:X43 was inserted

into pTZ-18~ that had been linearized with AccI to create pTZ-

18u:thr with a unique XbuI site following the thr attenuator. A BamHI

fragment (131 base pairs) from pCOS-54 (9, lo), which contains the

rrnC terminator, was cloned into pTZ-19u (7) to produce pTZ-

lSu:rrnC, which has a T7 promotor with a downstream rrnC termi-

nator. A HindIII-Xbal fragment (126 base pairs) from pTZ-18u:thr

was cloned between the Hind111 and XbaI sites of pTZ-19u:rrnC to

produce pTZ-19tt.

A nested set of deletions within the run of continuous template

deoxyadenosines in the thr attenuator was constructed previously in

pKB2000 plasmid derivatives (12). XbaI-MEuI fragments (47-54 base

pairs) from the pKB2000 variants were inserted between the MZuI

and XbaI sit,es of pTZ-19tt to produce the pTZ-19T series used in the

transcription assays (Fig. 1A).

The construction of single nucleotide and double nucleotide var-

iants in the thr attenuator was as follows (Fie. 1B). A HindIII-XbaI

fragment (127 base pairs) from pTZ-19tt was cloned into pTZ-19u to

form pTZ-19thr containing the wild-type thr attenuator. The var-

iants, BClO, ADl, BD16, which were made by cassette mutagenesis

(13), are in M13mp9 (3-5), and the other variants were constructed

in M13mplO (15, Table I). A Tug1 fragment (433 base pairs) from

these Ml3 derivatives was inserted into the AccI site of pTZ-18~ to

create a unique XbuI site following the thr attenuator. MluI-XbuI

fragments (60 base pairs) from pTZ-18~ containing the thr attenuator













Vectors and plusmids used in these studies

variants were cloned into the MluI and XbuI sites of pTZ-19thr to

generate the pTZ-19thr variants used in the transcription assays.

In Vitro Transcription with T7 RNA Polymerase-Transcription

reactions were similar to those described by Mead et al. (7). Reactions

(50 ~1) contained 40 mM Tris.HCl (pH = 8.0), 8 mM MgCls, 25 mM

NaCl, 5 mM dithiothreitol, 2 mM neutralized spermidine. (HCl)a, the

four unlabeled ribonucleoside triphosphates (1 mM ATP, UTP, and

GTP and 0.3 mM CTP), 2-6 &i of [w~‘P]CTP (final specific activity

= 0.13-0.4 mCi/nmol) and 0.5-1.0 pmol of DNA template. Mixtures

were incubated at 37 “C for 10 min before 2 units of T7 RNA

polymerase were added to initiate the reactions. After 50 min at 37 “C,

the reactions were brought to final concentrations of 10 mM

Na,EDTA and 0.2 mg/ml of carrier tRNA, extracted with phenol/

chloroform, l:l, and the nucleic acids precipitated with ethanol. The

pellets were dried under vacuum and-resuspended in 10 pl of 95%

formamide. 0.1% (w/v) xvlene cvanol. 0.1% (w/v) bromnhenol blue.

50 mM Tris. HCl, i4’mM H3B03,8nd 2.5 mM N&EDTA and analyzed

on 6% acrylamide, 8 M urea gels containing (TBE buffer) 50 mM

Tris-HCl, 44 mM HaBOa, 2.5 mM Na*EDTA as described by Maxam

and Gilbert (25).

Quantification of Termination Efficiency-Using the autoradiogram

as a reference, the bands corresponding to the terminated and read-

through transcripts were excised from the gel and quantified in a

scintillation counter. The data were corrected for background and

normalized according to the length and CMP composition of the

transcript. The background from the gel exclusive of the bands of

interest was from 2 to 4% of the total radioactivity in the lane. For

the pTZ-19tt series, the termination efficiencies of the thr attenuator

were calculated as (threonine-terminated band) x lOO/(threonine-

terminated band + rmC-terminated band + readthrough transcript

band), and similarly, that for the rrnC terminator as (rrnc-termi-

nated band) x lOO/(rrnC-terminated band + readthrough band). For

the pTZ-19thr series, the termination efficiencies of thr attenuator

were calculated as (threonine-terminated band) x lOO/(threonine-

terminated band + readthrough band). The results are reported as

means + standard deviations of at least four assays.

DNA Sequence Analysis-Since the pTZ-19u constructs contain

the bacterionhage fl orizin of DNA renlication. single strand DNA

from pTZ-l&t and pTZ-?9thr can be produced by infecting cells with

the helper bacteriophage MI3K07 (26). The resultant DNA was

sequenced by the chain-termination procedure of Sanger et al. (27)

using the large proteolytic fragment of DNA polymerase I. The double

strand DNA of some plasmids was similarly sequenced using modified

T7 DNA polymerase as described by Kraft et al. (28).


Termination at Rho-independent Terminators in Vitro-T7

RNA polymerase terminates at the thr and the rrnC termi-

nators. Cessation of transcription at these terminators is

shown in Fig. 3. The pTZ-19tt and pTZ-19thr plasmids were

linearized with MluI, X6a1, or EcoRI to produce templates

containing either no terminator, the thr attenuator, or both

the thr attenuator and the rrnC terminator, respectively (Fig.

2A), and these templates transcribed by T7 RNA polymerase

in vitro as described under “Experimental Procedures.” Since

the MuI site in plasmid pTZ-19tt is located before the thr

attenuator (Fig. 2A), DNA templates produced with this

enzyme direct the synthesis of a single transcript with the

Vectors Refs.

Contains a T7 promotor and the polylinker sites of pUC-19

Contains a T7 promotor and the polylinker sites of pUC-18

MluI-RAMHI fragment (43 base pairs)

Contains the rrnC terminator

Contains point variants in the G+C- and A+T-rich regions of the thr attenuator

(13IG, 138U, 140A, 151A, 153A, 160A, 16OC, 160G, 1316UA, 1316GC, and 156AC)

Contains nested deletions of the deoxyadenosine track (Tl to T8)

Contains point variants in the G+C region of the thr attenuator (ADl, BClO, BD16)

Contains the wild-type thr attenuator

Contains a T7 promotor, the thr attenuator and the rrnC terminator

Contains a T7 nromotor and the thr attenuator

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9, 10





This work

This work



M13mp9(3-6) Lambda phage pcos-54 plz19u

(rhr atpuator)


(rmC teiminator) (T7 prtmotor)


(thr attenuator)


(Deletions in dA track)

MU+ isolate

Xbal 54.47bp


T7 Hindlll

oromotor BamHl Mlul


(thr attenuator)


Hindlll I isolate

+Xbal 1 126bp ,

Rho-independent Termination of T7 RNA Polymerase 3825



(T7 promotor. thr

and rrn terminator)

Mlul +



attenuator Xbal


(T7 promotor and

rrnC terminator)





terminator BamHl EcoFll

+l 31 74


*... *...





134 270 291

..-- .-


. ..-


. ..-

. ..pTZ-19T


pTz-19tt pTz-19u MlBmplO and pTi!-1811

(thr and rrnC (T7 promotor) M13mp9(3-5)

terminators) (points variants

fin;ll (;;&l; ~ Iii;, 1 inT:;la[z; + Acci 1



(T7 promotor and (thr variants with Xbal site)

thr at enuator)

M/ul+ MU+ isolate

Xbal I

Xbal 60bp


promotor Hindlll Mlul



attenuator Xbal EcoRl


+l 74 134 159

*. .’



*. .*

‘. .*







pTZ-19thr variants *’

FIG. 1. Construction of DNA templates. A, scheme for the

construction of the pTZ-19T series containing successive deletions of

the stretch of template deoxyadenosines. B, scheme for the construc-

tion of plasmids containing point variants in the thr attenuator.

Transcription initiates at the T7 promotor in vitro at position +l,

and the numbers correspond to the lengths of the expected transcripts.

size expected for run-off transcription (Fig. 3, lane a). Simi-

larly, the template produced with XbaI, which is located

between the two terminators, shows one transcript arising

A) pTZ-19tt

T7 thr rrnc

promotor M/ill attenuator Xbal terminator EcoR I

Template: I




+I 74 134 291

Treated with Mlul B 74


Treated with Xbal

Treated with EcoRl

6) pTZ-19thr

T7 thr


promotor M/II


attenuator Xbal EcoRl



+l 74 134



Treated with Xbal 134

thr FIT

Treated wth EcoRl

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123 134

thr RT

123 226 291

thr ,rllC RT



lhr RT

FIG. 2. Schematic representation of templates and tran-

scripts. Only the T7 promotor and the polylinker regions containing

the terminator specifying DNA of pTZ-19tt and pTZ-19thr are shown.

The positions of the T7 promotor and the terminators are indicated

as thicker lines on the DNA templates. Transcription initiates at the

T7 promotor in vitro at position +l, and the numbers correspond to

the lengths of the expected transcripts. The positions of the tran-

scripts terminating at thr attenuator (t/w), rrnC terminator (rrnC),

and the end of molecule (RT) are indicated. The thickness of the

lines indicates the relative amounts of transcripts. A, the pTZ-19tt

plasmid contains the T7 promotor, the thr attenuator, and the rrnC

terminator. Deletion variants in the continuous stretch of deoxyaden-

osines were constructed in this plasmid (Fig. 1A). B, the plasmid

pTZ-19thr contains the T7 promotor and the thr attenuator. All point

variants in the thr attenuator were cloned into this plasmid (Fig. MI).

from the thr attenuator and another read-through transcript

(Fig. 3, lane b). The plasmid linearized with EcoRI and con-

taining both terminators produces the expected three tran-

scripts, two from the terminators and one from read-through

transcription (Fig. 3, lane c). The templates linearized by

either EcoRI or XbuI both contain the thr attenuator and

produce transcripts of equal size from this terminator al-

though the distances from the T7 promotor to the end of the

templates are of different total lengths and form different

length run-off transcripts (Fig. 3, lanes b and c). The efficien-

cies of termination by T7 polymerase on these plamids in this

experiment were 35 and 37%, respectively. The values from

this assay are smaller than the result of 43 + 3% reported in

Table II and illustrate the day-to-day variability of the assay.

Lanes d and e of Fig. 3 show similar results when another

template, pTZ-19thr, containing the thr attenuator on a

shorter molecule (Fig. 2B) is transcribed. Gels containing

RNA standards indicated that the sizes of the transcripts

produced in these experiments correspond well to expecta-

tions (data not shown). Collectively, these results show that

T7 RNA polymerase terminates at these two E. coli rho-

independent terminators with efficiencies of 35 and 32% at

the thr and rrnC terminators, respectively. Experiments with

supercoiled substrates indicate little effect of negative torsion

of the template on the termination at either the thr or rrnC

terminators by T7 RNA polymerase (data not shown). These

observations are consistent with the lack of significant super-

coiling effects detected with E. coli RNA polymerase in vivo

(29) and in vitro (30, 31).

Termination with Variants of the G+C-rich Region-Pre-

vious studies have shown that some point variants in the

G+C-rich region of the thr attenuator decrease transcription

termination by E. coli RNA polymerase (11-16). To test

3826 Rho-independent Termination of T7 RNA Polymerase

abed e

FIG. 3. Autoradiograph of the RNA transcription products

from pTZ-19tt and pTZ-19thr templates. The plasmids pTZ-

19tt and pTZ-19thr were linearized with the appropriate restriction

enzymes, transcribed wit.h T7 RNA polymerase in uitro, and the

transcription products analyzed as described under “Experimental

Procedures.” Plasmid pTZ-19tt contains both the thr attenuator and

the rrnC terminator (Fig. 2A). Plasmid pTZ-19thr has only the thr

attenuator (Fig. 2R). The readthrough transcripts terminating at the

MluI, XbaI, or EcoRI termini are marked. The transcripts terminating

at the thr attenuator and rrnC terminator are indicated as thr and

rrnC. Lane a, pTZ-19tt cut with MM; lane b, pTZ-19tt cut with XbaI;

lane c, pTZ-19tt cut with EcoRI; lane d, pTZ-19thr cut with XbaI;

lane e, pTZ-19thr cut with EcoRI.



Termination efficiencies of thr attenuator variants


Position Base pair 1\G Transcription

altered” change value’ termination’

kcal/mol %

Wild type -23 43 f 3

138U 100 CG+JJG -21 14 f 1

140A 102 CG-+AG -17 6-cl

151A 113 CG+CA -17 7+1

153A 115 CG-KA -16 13 -c 1

131G 93 AU+GU -22 43 + 4

160A 122 AU-AA -21 20 f 1

160C 122 AU-AC -21 132 1

160G 122 ALLAG -21 13 f 1

1316UA 122 & 93 AU-LJA -23 19 f 1

1316GC 122 & 93 AU+GC -25 13 + 2

156AC 115 & 122 CG+CA

AU+AC -14 4r1

AD1 117 CG+CC -16 8fl

BClO 98 CG+GG -16 522

BD16 98 & 117 CG-+GC -23 18 r 2

’ The positions correspond to those in Fig. 5.

’ AG values (kcal/mol at 37 “C) were calculated according to Freier

et al. (32).

Transcription termination was analyzed using the pTZ-19thr

derivative templates that had been linearized with EcoRI as described

under “Experimental Procedures.”

transcription termination by T7 RNA polymerase on altered

thr attenuators, variants containing the transcription termi-

nation structure of the threonine operon regulatory site (Fig.

5) were cloned into pTZ-19u. The variant DNAs were line-

arized with EcoRI and used as templates. The labeled tran-

scripts were analyzed as described under “Experimental Pro-

cedures,” and the results are shown in Fig. 4. The RNAs

synthesized from the variant templates migrated differently

than the transcript formed by templates with the wild-type

sequence. This mobility anomaly was previously reported (12,

15) and is likely due to the different stabilities of the variant

hairpin structures during electrophoresis.

The efficiencies of transcription termination by T7 RNA

polymerase on altered attenuators are presented in Table II.

abc d e f 9 hi j k I mno

FIG. 4. Autoradiograph of the in vitro transcription prod-

ucts from variants of the thr attenuator. The transcripts were

prepared and analyzed as described under “Experimental Proce-

dures.” The templates were pTZ-19thr derivatives linearized with

EcoRI (see Fig. 28). The terminated transcripts (thr) and the read-

through transcripts (RT) are indicated. Lane a, pTZ-19thr (wild type);

lane b, 13lG; lane c, 138U; lane d, 140A; lane e, 151A; lane f, 153A;

lane g, 160A; lane h, 16OC; lane i, 160G; lane j, 1316UA; lane k,

1316GC; lane 1, 156AC; lane m, ADl; lane n, BC 10; lane o, BD 16.


u c

Variant 1054 A-110 Variant



140A AG-CG+CA 151A


136U U G--C G-C A 153A


ED16 E 22 E-4


c A01

s6.A U.119

AU AA 160A







A C m(153A)




5’ 3’



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FIG. 5. The thr attenuator RNA and its variants. The sec-

ondary structure of the wild-type thr attenuator is presented as the

conformation that maximizes potential base pairing. The RNA num-

bering system is same as in Table II, and the nucleotide positions are

relative to the transcription initiation site (+l) of T7 RNA polym-

erase. The changed bases in each variant are indicated as bold letters

and shown in Table II.

The single-position variants in the G+C-rich region decrease

the frequency of termination from 43% with the wild-type

attenuator to from 14 to 6%. Four mismatches (140A, BClO,

151A, and ADl) show dramatic decreases in termination (4-

7%, Table II) suggesting that helix integrity is necessary at

these positions. Helix stability values calculated by the

method of Freier et al. (32) indicate that these variants are

decreased in stability by 5-28% with respect to the wild-type

structure. There is no direct correlation with the decrease in

termination efficiency and the decrease in helix stability, an

effect noted previously with the bacterial polymerase on these

same variants (11).

The C at position 100 forms a base pair with the G at

position 115 in the RNA secondary structure (Fig. 5). Variant

153A converts this CG base pair to a CA mismatch and gives

rise to 13% termination efficiency. Changing the C at position

100 to U to give a UG apposition (138U) shows a quantita-

tively similar termination value (14%). The UG position is

not a mismatch and can form a base pair (32). The predicted

stabilities of the two hairpins, 153A and 138U, differ by 4.6

kcal/mol. Their similar termination efficiencies suggest that

factors beyond simple helix integrity, for example, the se-

quence itself, are involved in the termination. Similar effects

have been observed with E. coli RNA polymerase (12).

Two different mismatches (140A and 151A) at the base pair

between positions 102 and 113 yield similarly decreased ter-

mination values. Since disruption (BClO and ADl) of the

base pair between positions 98 and 117 by two different

mismatches also drastically decreases termination, helix in-

tegrity at both ends of the G+C rich portion of the hairpin

stem are important. This conclusion is supported by the

finding that the compensating variant BD16 that forms the

inverted GC base pair, with respect to wild type, increases the

termination from approximately 7% in the mismatches to

18%. However, since the wild type with a CG base pair

terminates 43% of the time, this result again implicates fac-

tors in addition to helix integrity in the process.

Conversion of the base pair between position 100 and 115,

a site in the middle of the hairpin, to two different mismatches

(138U and 153A) decreases the termination to approximately

13% in both cases. The smaller effects of the mismatch in the

middle of the stem in contrast to those at its ends, even

though the predicted decreases in stability are similar (Table

II), again illustrates the lack of direct correlation between

helix stability and termination.

As mentioned above, the compensating variant BD16

changes CG to GC at positions 98 and 117 (Table II). The

calculated stability values for this variant and the wild-type

are -21.4 kcal/mol but the termination efficiencies are 18 and

43%, respectively. This variant vividly illustrates that se-

quence per se can affect the termination efficiency of T7 RNA

polymerase. Finally, the predicted stabilities of variants BClO

and 153A are approximately equal, but their termination

efficiencies differ by more than 2-fold.

Termination with Variants of the A+T-rich Region-To

determine the effects of changes in the sequence of the A+T-

rich region of the attenuator, variants which encode both the

runs of A and U residues in the transcript were tested. Some

of these variants disrupted each stretch individually whereas

others altered both sequences but left them capable of forming

a potential helix between the two regions (Fig. 5). Disrupting

the U tract (160A, 16OC, or 160G) by substituting A, C, or G

for U at position 122 decreases termination to 50% or less of

the wild-type value (Table II). The formation of uniquely

unstable dArU base pairs (33) between the template and this

region of the transcript has been assigned a role in factor-

independent termination with E. coli RNA polymerase (34,

35). The substitution of any nucleotide that encoded a base

other than U would give rise to a more stable template-

transcript base pair (33) and, according to the hypothesis, be

expected to decrease termination.

To test the effect of modifying the run of A residues

preceding the G+C-rich hairpin in the terminator transcript,

several variants were assayed. Alterations that change both

the A and U stretches to allow potential base pairing (1316UA

and 1316GC) both decrease termination. The similar extent

to which each does so, however, compared with the effect of

the identical change in the U run only, indicate that it is the

U stretch that is determinant. Variant 160A changes U to A,

as does 1316UA, and although the former change creates a

mismatch, the latter creates a potential inverted UA base

pair. The termination values for the two variants are nearly

identical (20%). Likewise, variant 16OC, which disrupts the

run of Us and creates a mismatch terminates with the same

efficiency as does the double variant 1316GC. Changing the

A at position 93 in the run of As (13lG) has no effect on

Rho-independent Termination of T7 RNA Polymerase 3827

termination. Collectively, these results suggest a dominant

role for the U tract, with respect to the A stretch, in termi-

nation. They further suggest that base pairing between the

two tracts is not crucial to termination.

An AC mismatch in either the G+C-rich region (153A) or

the U stretch (160C) decreases termination to one-third the

value of the wild type. The variant (156AC) containing both

these mismatches decreases termination to one-tenth the

wild-type value suggesting that the two regions independently

contribute to termination.

Effects of Varying the Length of the Deoxyadenosine Tract-

Lynn et al. (12) showed that the length of the deoxyadenosine

tract of the template encoding the stretch of uridines that

comprise the 3’ end of the transcript affects the termination

efficiency. We have tested these variants of the thr attenuator

in a series of plasmids (pTZ-19T, Fig. 1A) that also contains

the rrnC terminator immediately downstream as an internal

control for factor-independent termination. Both the wild-

type thr attenuator and the rrnC terminator stop transcrip-

tion with approximately the efficiencies described in the ear-

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abed ef gh

I - _ . . _- - - -



4Blmec thr

FIG. 6. Autoradiograph of products from templates with

deoxyadenosine deletions. Transcription reactions used the pTZ-

19t.t derivatives linearized with EcoRI as templates (Fig. 2A). The

position of the terminated transcripts of the thr attenuator (thr), the

downstream rrnC terminator (rrnC), and the readthrough transcript

(RT) are indicated. Lane a, pTz-19Tl; lane b, pTZ-19T2; lane c, pTZ-

19T3; lane d, pTZ-19T4; lane e, pTZ-19T5; lane f, pTZ-19T6; lane g,

pTZ-19T8; lane h, pTZ-19tt.



012345678 9 10

Number of Deoxyadenosines

FIG. 7. Transcription termination by T7 RNA polymerase

as a function of template deoxyadenosine residues. The termi-

nation efficiencies of the thr attenuator deletions and the rrnC

terminator in the pTZ-19T series are indicated with the closed (0)

and open (0) circles, respectively. The termination efficiencies of the

wild-type thr attenuator with 9 uridines and the rrnC terminator in

pTZ-19tt are indicated by the closed (A) and open (A) triangle,

respectively. The data are presented as means f standard deviations.

3828 Rho-independent Termination of T7 RNA Polymerase

lier experiment (Fig. 3). The results of successively shortening

the run of deoxyadenosines in the attenuator template is

shown in Figs. 6 and 7. There is a background level of

termination of about l-2% until it is possible to form a run

of 5 uridines, at which point termination rises to 5%. When

a stretch of 8 uridines can be formed the termination is nearly

the wild-type value. In all these variants, the termination

efficiency of the rrnC terminator remains essentially constant

at 29-32%. These results show that T7 RNA polymerase

needs an attenuator template with at least 5 deoxyadenosine

residues to begin to cease synthesis, and that 8 or 9 are

required for maximal termination.


Both a G+C-rich region of dyad symmetry and an A+T-

rich segment of the DNA encoding rho-independent tran-

scription-termination structures are required for efficient ces-

sation of RNA synthesis by E. coli RNA polymerase (34-39).

We find that both the stability and the sequence of the RNA

hairpin formed by G+C-rich region and also the length of

uridine track influence the termination of T7 RNA polymer-


In the thr attenuator there are at least three sequence

segments that might affect termination efficiency with this

terminator, the G+C-rich hairpin, the uridine stretch, and an

adenosine stretch preceding the hairpin which is potentially

complementary to the uridine run. Additionally, since se-

quences both preceding and following these structural ele-

ments affect termination by E. coli RNA polymerase (30, 31,

40,41) they might also influence the bacteriophage enzyme.

Five single changes in the G+C-rich hairpin (140A, 15lA,

153A, ADl, and BClO) each create a mismatch, destabilize

the helix, and decrease termination (Table II), and three of

these variants, BClO, ADl, and BD16 exist in a slightly

different sequence context than do the remainder of the

constructions. In these three variants the template deoxy-

adenosine stretch is 8, and not 9, residues long as it is in the

wild-type thr attenuator. Since the termination efficiencies of

E. coli RNA polymerase are the same with attenuators con-

taining eight or nine template deoxyadenosines (12, 13) and

do not change greatly with T7 RNA polymerase (this work),

the effects we observed with these variants are Iikely due to

the changes in the G+C-rich hairpin and not to the number

of deoxyadenosines.

Brendel et al. (42) have reported a consensus sequence,

CGGG(C/G), in the G+C-rich region of procaryotic rho-

independent terminators. This sequence is present in the thr

attenuator, and we find that altering it wituout disrupting the

base pairs within it (BD16 and 138U) decreases termination.

Thus, variant BD16 converts a CG pair to a GC pair, and

variant 138U converts a CG pair to a UG pair, changes which

should not destabilize the helix greatly, and termination is

decreased. These results suggest the helix sequence per se is

involved, with the caveat that the algorithms used to calculate

helix stability may not be completely adequate.

Variants in the G+C-rich region led to transcripts with

anomalies in their electrophoreitc migration. These anomalies

have been attributed to the different stabilities of the RNA

helices of the variants (12). There is not a perfect correlation

between the calculated stabilities and the relative mobilities,

but some systematic relationships exist. Variants in the G+C-

rich region that disrupt the helix (140A, 151A, 153A, ADl,

BClO, and 156AC) migrate more slowly than the wild-type

helix. If the variants are in the A+U-rich region (160A, 16OC,

and 160G), their mobilities are similar to the wild-type helix

(Fig. 4). For variants that do not create mismatches in the

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helix, the calculated free energies of melting correlates with

mobilities of the transcripts. Thus, the predicted stabilities of

the variants without mismatches are, listed from most stable

to least stable: 1316GC > 1316UA = BD16 = wild type >

138U, and the same order is found for the relative mobilities

of these variants during electrophoresis (Fig. 4). These results

suggest that the transcripts are not totally denatured and that

the various conformations of the RNAs are in rapid equilib-

rium during electrophoresis.

E. coli RNA polymerase has been studied with many of

these same variants (12, 13, 15). Alterations in the G+C-rich

region showed less dramatic effects with the bacterial enzyme

than we observed with T7 RNA polymerase. The bacterial

enzyme terminates more efficiently with the wild-type atten-

uator than does T7 polymerase (90 versus 43%), and the

variants have a less pronounced effect with a maximum

decrease to 70% termination. The studies with E. coli RNA

polymerase were performed in 150 mM KC1 (12, 13, 15), and

those with the T7 enzyme in 25 mM NaCl (“Experimental

Procedures”). Increasing the ionic strength to 150 mM NaCl

or 150 mM potassium glutamate increases the termination

efficiency of T7 RNA polymerase to 60%. However, the tran-

script decreases 3.5-fold in amount. Thus, at comparable ionic

strengths, T7 RNA polymerase fails to terminate as efficiently

as the bacterial enzyme at this attenuator. When we at-

tempted to compare the two enzymes at the 100 PM rNTP

concentrations usually used in studies of E. coli RNA polym-

erase (12, 13, 15), T7 RNA polymerase formed less than 3%

of the amount of RNA made under our usual conditions (1

mM rNTP). The termination efficiency of T7 RNA polymer-

ase increased from 40% to approximately 80% as the rNTP

concentration was decreased through this range. We conclude

that under conditions optimized for RNA synthesis, the T7

enzyme is less efficient in transcription termination of this

attenuator than the bacterial enzyme, but that, as is the case

with E. coli RNA polymerase (30), the termination efficiency

of the bacteriophage enzyme increases as the rNTP concen-

tration decreases.

When the nucleotide analogue ITP was substituted for GTP

the variants led to dramatically decreased terminations with

the bacterial enzyme (43, 44). Substitution of inosine for

guanosine destabilizies RNA helices (45). Since T7 RNA

polymerase responded to disruptions in the G+C-rich region

without the amplification due to inosine incorporation, it

probably requires a more perfect and thus more stable hairpin

for effective termination than does the bacterial enzyme.

The A+T-rich sequences encode a stretch of uridine resi-

dues that may lead to a destabilized transcript-template com-

plex (12, 46). In the thr attenuator the uridine stretch is

capable of forming an intramolecular duplex with an A-rich

region preceding the G+C-rich hairpin, i.e. of extending the

base of G+C-rich hairpin by forming AU base pairs (Fig. 5).

Since fewer variants were analyzed in this region than within

the hairpin, less can be said concerning the role of the A-rich

region. Variant 13lG converts an AU to a GU with no effect

on termination. The other variants in the A stretch, 1316UA

and 1316GC, simultaneously alter both the A and U runs,

making the observed effects difficult to assign solely to the A

region. The stretch of adenosines preceding the G+C-rich

stem of the thr attenuator allows the sequence to function as

a terminator when cloned in an inverted orientation.’ When

the attenuator containing the set of nested deletions is cloned

in the reverse orientation a terminator template is formed

with a decreasing number of adenosines in front of the hairpin

1 S.-T. Jeng, J. F. Gardner, and R. I. Gumport, unpublished obser-


and a constant potential 6 uridines following it. Results from

such constructions indicate that varying the number of adenosines

has much less affect on termination than varying the

number of potential uridines after the G+C-rich hairpin in

the usual orientation (data not shown). Taken together, these

results suggest that the extension of the G+C-rich hairpin

through the formation of A-U base pairs in the transcript is

not essential for termination by T7 RNA polymerase.

The 3’ ends of the transcripts produced by T7 RNA polymerase

at the thr attenuator were determined by RNase Tl

digestion followed by electrophoresis (data not shown). Compared

with the E. coli RNA polymerase, which terminates

predominately at the seventh and eight template deoxyadenosine

following the G+C-rich region (14), the majority of the

T7 RNA polymerase transcripts terminate at the fifth, sixth,

and seventh deoxyadenosines.

The nested deletion variants exist in a slightly different

sequence context than does the wild-type construction. In all

of the nested deletion variants, the sequence immediately

following the template deoxyadenosine stretch had the five

deoxynucleotides, GACTC, deleted as a result of the cloning

procedures. Thus, compared with the wild-type sequence of

pTZ-19thr, . . .CGACTCTAGA.. ., the sequence of these

nested deletion variants is . . .CTAGA.. . . Since the termination

efficiencies of E. coli RNA polymerase are the same with

attenuators containing eight (one of the nested deletion variants)

or nine deoxyadenosines (12, 13) and do not change

greatly with T7 RNA polymerase (Fig. 7), the effects observed

with the nested deletion variants are not likely to be due to

the changes in these five deoxynucleotides. Since these constructions

with eight deoxyadenosines show termination efficiencies

similar to those with the wild-type construct containing

nine deoxyadenosines, the sequences immediately distal

to the hairpin do not greatly affect termination under

these conditions. This result contrasts with those observed

with E. coli RNA polymerase on some T3 and T7 terminators

(30, 31). Telesnitsky and Chamberlin (31) reported that the

sequences between three and seven nucleotides downstream

of the T7Te terminator release site affect the strength of

terminator. When the sequences distal to sites releasing transcripts

with T7 RNA polymerase in the wild-type and the

nested deletion variants are comnared. four of the seven

nucleotides are identical

This identitv may exnlain


why we failed to observe downstream

sequence effects. Alternatively, T7 RNA polymerase

may not be affected by downstream sequences.

Single nucleotide changes in the U stretch (160A, 16OC,

and 160G) lead to decreased termination, supporting the

hypothesis that uridine residues uniquely destabilize the template-transcript

duplex and thereby contribute to termination

(33, 35). In addition, successive deletions of the A+T-rich

region so that the template encodes fewer uridines (Fig. 7)

also indicate the importance of this region in the termination

of T7 RNA polymerase. A similar, but less dramatic effect,

was first reported with E. coli RNA polymerase (12). Whereas

E. coli RNA polymerase terminates at 19% efficiency of the

wild-type value with 3 potential uridine residues in the transcript,

T7 RNA polymerase requires 6 uridine residues to

attain 15% of the wild-type termination efficiency. Maximum

termination requires more uridine residues with T7 polymerase

than with the bacterial enzyme.

Some other rho-independent terminators, e.g. the rrnBT1

(17, 18), rrnC terminator (this work), thr attenuator, and T$,

a T7 late terminator (20-22), can stop T7 RNA polymerase.

The rrnC terminator encodes a hairpin structure followed by

8 uridines, and the predicted free energy of melting is -9.6

Rho-independent Termination of T7 RNA Polymerase 3829

kcal/mol, a value which is much lower than that of thr

attenuator. Nevertheless, the termination efficiency of T7

RNA polymerase with the rrnC terminator is approximately

equal to that with the thr attenuator. The sequence TCTG is

found downstream of many terminators, and it has been

suggested that this motif may affect termination efficiency

(47-49). The rrnC terminator has this sequence, the thr

attenuator lacks it, and this difference may explain the equal

termination of the rrnC terminator in spite of its less stable

hairpin structure. Further experiments would be required to

test this hypothesis.

Te, an early terminator that is effective with E. coli RNA

polymerase does not affect T7 RNA polymerase (19-22). This

terminator has but three continuous deoxyadenosines follow-

ing G+C-rich region and thus may be unable to form sufficient

uridines in the transcript to cause efficient termination with

the phage enzyme. Furthermore, a template containing a run

of discontinuous deoxyadenosines lacking a preceding poten-

tial hairpin structure terminates T7 RNA polymerase with

90% efficiency (7). These results indicate that not all rho-

independent transcription structures in E. coli terminate T7

RNA polymerase and that there are other factors residing in

either the template or the transcript that can signal the

enzyme to cease synthesis.

We have found that when the thr attenuator forms a

transcript with an intact G+C-rich hairpin and can poten-

tially encode at least 8 uridines it terminates T7 RNA polym-

erase with maximum efficiency. Compared with the bacterial

enzyme, the bacteriophage enzyme requires a more perfect

hairpin and a longer uridine tract to terminate. In addition,

the absolute value of the maximal termination is less with the

T7 polymerase than with the bacterial enzyme with this

terminator under the usual in vitro reaction conditions used

for each of the enzymes. We are presently determining the

termination efficiencies in uiuo of some of the thr attenuator

variants with these two enzymes to examine their relevance

under physiological conditions.

Acknowledgments-We thank 0. C. Uhlenbeck, M.-T. Yang, and

H. Scott for valuable discussions. We thank B. Kemuer for his

generous gifts of the plasmids pTZ-19u and -18~ and E. Morgan for

his gift of the plasmid pCOS-54. We are grateful to R. Reynolds and

M. Chamberlin for providing results prior to their publication.












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