THE JOURNAL OP BIOLOGICAL CHEMlWRY 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*

(Received for publication, August 30, 1989)

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

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.

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 stabilities.

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.

3823

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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.

EXPERIMENTAL PROCEDURES

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 assays.

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

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).

RESULTS

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

TABLE I

Strains

Vectors and plusmids used in these studies Vectors Refs.

pTZ-19u pTZ-18u XC,875 pcos-54 Ml3mplO

pKB2000 M13mp9(3-5) m13mp9(3-6) pTZ-l&t nTZ-19thr

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

7

24 9, 10

12,15

12 13 11 This work This work

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Rho-independent Termination of T7 RNA Polymerase 3825

A)

M13mp9(3-6) Lambda phage pcos-54 plz19u (rhr atpuator) I

(rmC teiminator) (T7 prtmotor)

M13mpk143 (thr attenuator)

pTZ-l&l

pTi!-18u:thr (thr attenuator)

Hindlll isolate I ] +Xbal 1 126bp ,

plZl9u:rrnC (T7 promotor and

rrnC terminator)

Hindill +Xbal

pKB2000 (Deletions in dA track)

MU+ isolate

I Xbal 54.47bp

+ pTz-19tt

(T7 promotor. thr and rrn terminator)

Mlul + Xbal

T7 Hindlll oromotor BamHl Mlul

thr attenuator Xbal

rrnC terminator BamHl EcoFll

+l 31 74 134 270 291 --..

*... ..--- ..-- .-

*... a... . ..-

--.. *...

. ..- . ..-

..-- -...

pTZ-19T series”

8)

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

pTZ-19thr pTZ-18u:varlants (T7 promotor and (thr variants with Xbal site)

thr at enuator)

M/ul+ MU+ isolate Xbal I Xbal 60bp

T7 promotor Hindlll Mlul

I

thr 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 I I

Transcripts: +I 74 134 291

Treated with Mlul B 74 RT

Treated with Xbal 123

134 thr RT 123 226 291

Treated with EcoRl thr ,rllC RT

6) pTZ-19thr T7 thr promotor M/II attenuator Xbal EcoRl

Template: I I I I

+l 74 134 159 Transcript:

123 Treated with Xbal 134

thr FIT

123 Treated wth EcoRl 159

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

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3826 Rho-independent Termination of T7 RNA Polymerase

abed e

thr

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.

TABLE II Termination efficiencies of thr attenuator variants

Variant 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.

GA u c

Variant 1054 A-110 Variant CG AU

140A AG-CG+CA 151A GC

136U U G--C G-C A 153A SC10 ED16 E 22 E-4 c A01

GC s6.A U.119

AU AA 160A

131G GU AU AC 1316UA

1316GC

UA>AU AG 4

16OC

160G

GC AU A C m(153A) 156AC

CACAG A U UUUCGACTC 5’ 3’

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

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Rho-independent Termination of T7 RNA Polymerase 3827

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

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-

abed ef gh I - _ . . _- - - -

RT rrnC

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.

Lt”

0 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.

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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.

DISCUSSION

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- ase.

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

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- vations.

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Rho-independent Termination of T7 RNA Polymerase 3829

and a constant potential 6 uridines following it. Results from such constructions indicate that varying the number of aden- osines 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 polym- erase at the thr attenuator were determined by RNase Tl digestion followed by electrophoresis (data not shown). Com- pared with the E. coli RNA polymerase, which terminates predominately at the seventh and eight template deoxyaden- osine 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 termina- tion efficiencies of E. coli RNA polymerase are the same with attenuators containing eight (one of the nested deletion var- iants) 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 con- structions with eight deoxyadenosines show termination ef- ficiencies similar to those with the wild-type construct con- taining nine deoxyadenosines, the sequences immediately dis- tal 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 tran- scripts with T7 RNA polymerase in the wild-type and the nested deletion variants are comnared. four of the seven nucleotides are identical (TTCGACT &XL.S TTCTAGA). -- -- This identitv may exnlain why we failed to observe down- stream 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 tem- plate-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 tran- script, T7 RNA polymerase requires 6 uridine residues to attain 15% of the wild-type termination efficiency. Maximum termination requires more uridine residues with T7 polymer- ase 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

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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