A possible termination factor for transcription in Escherichia coli

Vol.

56,

No.

3, 1974

BIOCHEMICAL

A POSSIBLE

Department New

:Vovember

BIOPHYSICAL

TERMINATION FACTOR FOR IN ESCHERICHIA COLI

Huey-Lang

Received

AND

Yang

and

RESEARCH

COMMUNICATIONS

TRANSCRIPTION

Geoffrey

Zubay

of Biological Columbia University York City, New York

Sciences 10027

26,19i'3

SUMMARY: DNA from both Xdlac and X lac viruses direct mRNA-mediated synthesis of B-galactosidase (B-gal) a coup + ed cell-free system. When Adlac ONA is used the system is almost completely dependent upon the presence ofdenosine 3’,5’-cyclic monophosphate (CAMP) which is required for normal initiation of transcription at the lac promoter. By contrast when Aplac DNA is used about half the maximum amountof B-gal is synthesized in the absence of CAMP. It seems highly likely that this CAMP independent synthesis of B-gal is the result of read-through from a phage initiation site on the X@ DNA. Since there is believed to be at least one transcription termination signal before the lac operon it must be asked why this signal is not being recognized as such7 vitro. We hypothesize that the normal bacterial termination factor is missing or inactive in the bacterial extract used to make the cell-free system. This view is supported by the finding that protein extract can be isolated from whole &. coli cells under mild conditions which prevents the CAMP-independent R-gal synthesis without inhibiting CAMP-dependent B-gal synthesis. The behavior of the active factor (called the py factor) in this extract is different from either the Roberts p factor (called oR here) or the Schafer-Zill ig K factor. Furthermore it has been found that the pR factor cannot replace the py factor. Two

events

elongation

are

process

has

been

firmly

must

be

released.

more

than

by

at

from

one

cent

down

other

than

bacterial for

loci

on In

genes

termination

the

the

E. (as

which

in

i RNA

we

studies

will

0 I9 74 by Academic Press, Inc. in any form reserved.

of reproduction

of of

in

T7

bacteriophage

early

is

a

at

terminated without

genes)

tenta,tlvely

protein

the

a

may call

there

Thus

it

the

been termi-

called

p discovered

transcription

begins

point

about

20

obtained

evidence

py

may

DNA

of

require

which

has

involvement

We have

(2).

that

X bacteriophage

presence

725 Copyright AN rights

transcription.

chain,

elongation,

suggests

the

phage

RNA during

in

polymerase

The

synthesized

transcription

chromosome

to

transcription.

complex

of

and

opposed

newly

vitro

termination

DNA

the

the

of

polymerase

from

contrast

of co1

and

early

the

termination

DNA-RNA

chromosome

length

the

halted

for

so-called

of

the

to

that

(1).

the

be

mechanism

twlo

end

to

Evidence

one

Roberts

must held

demonstrated nates

essential

yet factor

any

another

(3,

per proteins that protein

4).

For

be

Vol.

56, No.

3, 1974

purposes

of

BIOCHEMICAL

distinction

the

AND

Roberts

p

BIOPHYSICAL

factor

RESEARCH

will

be

COMMUNICATIONS

referred

to

as

the

pR

factor. MATERIALS

AND METHODS: Conditions for cell-free synthesis and assay. For mcell-free synthesis of the enzyme B-galactosidase the S-30 extracts used were prepared from strain SB 7223 (5) containing a deletion of the entire lac region. Either Xe DNA or he DNA were used for B-gal synthesis. Kept for slight modifications, all procedures used for synthesis, enzyme assay, and preparation of bacterial S-30 extracts and DNA have been described in detail elsewhere (6). The incubation mixture contained per ml: 44 un-ol Tris-acetate, pH 8.2; 1.37 ymol dithiothreitol (DTT); 55 umol KAc; 27 urn01 NH4Ac; 14.7 pm01 Mg(Ac)2; 7.4 umol Ca(Ac)2; 0.22 umole amino-acids; 2.2 umol ATP; 0.55 umol each GTP, CTP, UTP; 21 umol phosphoenolpyruvic acid; 100 ug tRNA; 27 ug pyridoxine HCI; 27 ug triphosphopyridine nucleotide; 27 pg flavine adenine dinucleotide; 11 ug p-amino-benzoic acid; 0.5 umol CAMP. These were preincubated for 3 min. at 37°C with 100 y/ml of Adlac or Xplac DNA with shaking before 9.0 mg S-30 protein extract was added. incubation with shaking was continued for 60 min. at 37°C. After synthesis, a 0.2 ml aliquot was removed and assayed for B-gal with 0-nitrophenyl B-D-galactoside. Enzyme assays were done at 29“C. Isolation procedures used for p factor. As a rule strain SB 7219 (5) containing a deletion of the entire Y ac region was usually used for preparation of the py factor. Strain SB 72rand several other strains containing lac deletions were also found to be effective sources of py factor. Growth rbacteria and the preparation of S-100 fraction have been described (7). All preparations were carried out at 0-4°C. Samples were dialyzed against buffer I (0.01 M Tris-Ac pH 8.2, 0.014 M Mg(Ac)2, 0.06 M KAc, 0.35 mM DTT, 5% glycerol) before assaying their effect on B-galactosidase synthesis. Preparation of 'crude DEAE extract'. After overnight dialysis in buffer II (0.01 M KH2P04-KOH pH 7.7, 0.35 mM DTT, 5% glycerol), the S-100 fraction prepared from 50 g of cells were passed through a 120 ml DEAE-cellulose, 0.91 meq/ml., pre-equilibrated in buffer II. After rinsing the column with 3 volumes of buffer II, the DEAE-cellulose bound protein was eluted with buffer II containing 0.3MNaCland precipitated by adding two volumes of cold saturated (NH4)2 SO4 to the eluate. The resulting precipitate was collected by centrifugation at 10,000 rpm in a Serval GSA rotor and resuspended in 25 ml of the appropriate buffer for further purification. This DEAE-cellulose preparation is called the 'crude DEAE extract'. It has an O.D. 2801260 ratio greater than 1.4 which indicates that all except traces of nucleic acid have been eliminated. DNA-cellulose column purification step. Double stranded calf thymus DNA-cellulose was prepared as described by Alberts (8). The DNA-cellulose prepared contained 1600 ug DNA/ml of column volume. The column was equilibrated in buffer IV (0.01 M KH2PO4-KOH pH 6.4, 0.35.mM DTT and 5% glycerol). 10 ml of DNA-cellulose was used for each 50 g of cells. After rinsing the column with buffer IV the py activity was eluted with buffer IV containing 0.3 M NaCl. --RESULTS

AND

cell-free upon B-gal Xdlac, absence

DISCUSSION:

synthesis the is 50 of

It of

of

CAMP

obtained

in

the

80

CAMP.

per

cent The

been

reported

B-galactosidase

presence

to

has

(9);

of same

(B-gal) only

absence the lac

that

2 to of

IO

CAMP.

optimal promoter-operator

726

is per

Xplac of

)\dlac

almost

cent

When level

the

B-gal region

DNA-directed

completely

of

the DNA is is

dependent

optimal is

used

level instead

of of

obtained

in

the

present

in

the

Vol.

56,

two

viral

some.

No.

3, 1974

DNAs In

a

In

- lac

lac

to

obtain

the

b2

The

i gene

1

presence

site

of to

thing required

role s-30. polarity

is

a

termination

the

of

for

i,

DEAE

to

Figure

I).

region genome region

b2

If

the

A possible transcription

DNA

initiation

at

a

X

that

phage

proteins,

(CAP),

(11). is

chromo-

two

with

region

reads

the

the

is

through

lac

promoter

is

The

(13).

the

S-30

extract’

was in

i

lac

gene

should

be

inactive

indeed

the

ara

C regulatory

idea

that

bacterial

materials

by

adding and

and

are

methods)

727

unless

absent

other

some-

known

in

to

the

S-30 playing

be (5)

inactive and

extract S-30

immediately

a in

the

factor

protein the

A factor

proteins

termination

to

initiation

occur

protein

a crude

the

process.

or some

ref. 11). P, 0, Z and

promoter-operator

a phage

not

the

presented

implying

the

termination

might

tested

12)

from

transcription

been

to

(modified from i (partial) --lac

(IO,

process for

has

5

transcribing

synthesis.

case

adjacent

Evidence

the

into

termination

the

innnediately

in Xplac harbmg of A.

between one

or

right-to-left

elongation

cell-free

factor

‘crude

lac

close

transcription-translation

Such

in

on for

from

in

normal

the

(see

signal

malfunctioning

missing

noted

dependence

region

virus

protein

been b2

the only

activator

the

CAMP

very

operon.

right

used in

either

transcribed

lac

for

extract

contains

has

necessity

map of the bactex into the

gene

the is

total nearby

region

of

region

from other

is

The genetic line shows inserted

the

within

the

promoter-operator

that

inserted

of

COMMUNICATIONS

synthesis.

---lac

FIGURE 1. The heavy Y (partial)

regions

gene

is

RESEARCH

which

catabolite controls

eliminating

e-gal

different

normal

some

BIOPHYSICAL

system

the

escape

or

operon

into

from

operon

for in

inserted

and

escape

explanation

the

is

polymerase

tble

initiated

it

AND

transcriptional

partial

Aplac -

lac

but

purified

RNA similar

BIOCHEMICAL

the

SuA

might

be

(called

a prior

Vol.

56,

No.

3, 1974

BIOCHEMICAL

AND

BIOPHYSICAL

--TABLE Effect of Description of py containing extract added no

DEAE

DEAE extractd

extracte

b

c d e f

COMMUNICATIONS

1

and CAMP directed

B-ga 1 (- CAMP)

on the synthesis by X& DNA.a mg of protein ml of

PY activityb

py

containing added per incubation mix

2.20

1.05

2.09

1.95

0.13

15.0

5.76

2.61

0.06

43.5

5.76

2.52

0.13

19.4

1.3

1.26

0.135

9.33

0.44

extractlc

DNA-cellulose

a

B-gal (+ CAMP)

addition

‘crude

HA

of py factor B-galactosidase

RESEARCH

extractf

0

Synthesis and assay are carried out as described in materials and methods. B-gal is reported as A420 corrected to an assay time of 200 min. is defined as the ratio of S-gal synthesized with CAMP The ‘p activity’ to tha Y synthesized without CAMP added. For a given extract, concentration dependence studies have shown that the amount of active py factor is approximately proportional to the ‘py activity’. ‘Crude DEAE extract’ is described in materials and methods. DEAE extract is the peak fraction from a gradient elution on DEAE-cellulose as described in the caption to Figure 2. HA extract is a 0.18 - 0.35 M phosphate cut from a Hydroxyapatite column described in the caption to Figure 3. DNA-cellulose extract is the 0 to 0.3 M NaCl fraction from a double strand DNA-cellulose column as described in materials and methods.

to

cell-free

synthesis

the

CAMP-independent

out

appreciable efforts

which

inhibits

guide

the

extract

’

NaCl before extract’

on have

the

subjected

has

been

fractionated

has

also

The

active

body

of

been

Under

these

B-gal

is

directed read

to

of

elution

as to

shown

fractionation

728

as

the

py.

purification on

Table

inhibitory to

The

py in

on

as 2.

columns

with-

protein

factor

property

to

‘crude

DEAE

‘Crude column

elutes

Figure

that

1).

steps.

a DEAE-cellulose

containing

protein

(see

the

found

as

eliminated

of

referred

a variety

was

completely

purification using

is

fraction

it

synthesis

through

by

subjected

almost

at

factor

the

conditions,

CAMP-dependent

This

been

main

the

apparent

has

the

of

been

purification.

gradient.

B-gal.

synthesis effect

Recent

extract’

of

added

a

sharp

The

‘crude of

DEAE using

peak DEAE

hydroxyapatite

a

Vol.

56,

No.

3, 1974

BIOCHEMICAL

6-

9-

4-

6-

AND

4

8

BIOPHYSICAL

12 FRACTION

16 NUMBER

RESEARCH

20

COMMUMICATIONS

24

FIGURE 2. Gradient elution pattern on DEAE-cellulose. The ‘crude DEAE extract ’ (described in materials and methods) dialyzed in buffer II overnight was applied to a 60 ml DEAE-cellulose (Whatman DE-52, 1.5 x 33 cm) column pre-equilibrated in buffer II with a linear gradient of 0 to 0.5 M NaCl. Fractions of 5 ml were collected, dialyzed against buffer I and assayed. ‘p activity’ (‘p activity’ is defined in Table 1) elutes as a sharp peak argund 0.22 M NaCY.

and

double-stranded

(0.26

hydroxyapatite enrichment

is

the

DEAE

‘crude

About

1/6th

0.3M by

of

is

This

suggests

than

just

the

on not

retained the

phosphate

this binds

bound

suggests

that the

in

extract’

procedure

activity’

M phosphate)

achieved

NaCI.This this

The

DNA-cellulose. and

to

binding groups

is

Only

activity’

elutes

estimated

that

about

l/lOth

a DNA-cellulose

a maximum It

the

‘py

of

60-fold

is

of

even

to

DNA-cellulose

of

the

DNA.

729

of

The

late SO-fold

the in

on

protein

0.01

of

M phosphate.

activity’

is

released

enrichment

is

obtained

great

phosphocellulose

very about

column

containing

DNA-cellulose. by

it

step.

protein that

‘py

interest at

low

The

that ionic

by

‘py strength.

involves

something

enrichment

of

py

more by

the

above

Vol.

56,

No.

BIOCHEMICAL

3, 1974

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

- 0.6 7 -0.5

i : L

-0.2

=

-0.1

5 d I

b

0

0

4

8 12 FRACTION

16 20 NUMBER

24

28

-0

32

(BioFIGURE 3. Gradient elution pattern on hydroxyapatite. Hydroxyapatite gel HT) was pre-equil ibrated in buffer I II (0.01 M KH2P04-K2HPO/+ pH 6.8, 0.35 After overnight dialysis in buffer III, the ‘crude mM DDT and 5% glycerol). DEAE extract’ was loaded on a 32 ml column (1.5 x I8 cm.) and eluted with a 3.5 ml fractions are collected and linear gradient of 0 to 0.6 M phosphate. The peak of ‘py activity’ assayed for ‘py activity’ as described in Table 1. is found around 0.26 M phosphate.

described

procedures

the

specific

This

is

are

because

procedures active

were

overcome

be

effectively

of

pR

nation

and

the

has

(either

prepared

py

than

partially

in

this

would

occurs

problem

so to

be

purified

activity

combined

to

been

release that

various

hypothesized

drawn

that

indicate

the

greater

that achieve

from

fractions

during the

indicated

1).

purification.

various total

(Table

We

fractionaticn purification

of

a

factor.

immediately

factor

much

loss

to

py

view

of

appreciable

may

In

probably

activities

attempting

highly

is

of and by

the

idea

described early

pR us

function

are or

of

that by

we

factor

in

might

be

assaying

Roberts

transcription

in

different obtained

py

(I)

which

X.

However,

factors. from

N.

730

Thus tlinkley)

termination for

catalyses

the was

various

the

same

the

termi-

tests

Roberts found

we

to

pR

factor

be

ineffective

Vol.

in

56,

No.

3, 1974

preventing

the

affinity

main

On

peak

of

phosphocel

whereas

should

called The

,also K

which

elution

py

be

and

pR on

pR

is

is

not

pR

is

retained

and

that

Schafer

of

on

B-gal.

one

the

elutes

of

RNA

between 0.1

Zillig to

phosphocellulose

made

before to

pH

be

in at

isolated from

0.01 pH

DNA

it

from

(14). both

pR

PY. The may

be

potential involved

in

evidence

presented

eliminate

the

contribute

to

further

importance

so

that

termination is

only

possibility the

the of

py

may

for

factors

process.

ACKNOWLEDGEMENTS: We are grateful isolated hy the Roberts method. the National Institutes of Health thank Dr. M. Gefter for suggesting

described for

support other

properties

factor

transcription

indirect that

termination its

of

We are be

studied

here

active

role

and

in

the

S-30

greater

to N. Minkley for a This work was supported (2-ROl-GM-16648-04). the use of hydroxyapatite

that

genes.

a

purifying in

is

bacterial

such

the

py

does

it The not

extract

may

factor

depth. sample of p factor by a grant from We would like to in purification.

REFERENCES 1. 2.

3. 4. 65:

7. 8. 9. 10. 11. 12.

13. 14.

Roberts, J.W. (1969) Nature 224, 1168; Roberts, J.W. (1970) Cold Spring Harbor Symp. Quant. Biol. 35, 121. Millette, R.L., Trotter,. C.D., Herrlich, P., and Schweiger, M. (1970) Cold Spring Harbor Symp. Quant. Biol. 35, 135. Zubay, G., Yang, H-L., Reiness, G., and Cashel, M. (1973) k Molecular Cytogenetics, pp. 253-261, Plenum Pub. Corp. Zubay, G. (1973) Ann. Rev. Genetics 7, in press. Yang, H-L., and Zubay, G. (1973) Molec. Gen. Genet. 122, 131. Zubay, G., Chambers, D.A., and Cheong, L.C. (1970) in The Lactose Operon, Beckwith, J.R. and Zipser, D., eds., Cold Spring Harbor Laboratory, p. 375. Zubay, G., Morse, D.E., Schrenk, J., and Miller, J. (1972) Proc. Nat. Acad,. Sci. 69, 1100. 8.71971) in Methods in Enzymology, L. Grossman and K. Moldave, Al berts, eds., vol. 4, p. 19r Chambers, D.A. and Zubay, G. (1969) Proc. Nat. Acad. Sci. 63, 118. Eron, L. and Block, R. (1971) Proc. Nat. Acad. Sci. 68, 18%. Malamy, M.H., Fiandt, M., and Szybalski, W. (1972) Molec. Gen. Genet. llg, 207. Kuma r , S. and Szybalski, W. (1972) J. Mol. Biol. 4D, 145. Wetekan, W. and Ehring, R. (1973) Molec. Gen. Genet. l&, 359. Schafer, R. and Zillig, W. (1973) Eur. J. Biochem. u, 201.

731

7.0.

a protein

T5

distinguish

eluted.

6.4-7.0

M phosphate

have

be

are

elutes

proteins

around

and

py

last

retained

the

phosphocellulose

gradient

of

COMMUNICATIONS

Furthermore and

a salt

appreciably

pieces K

RESEARCH

DEAE-cellulose

py

shorter

properties

of

using

whereas

mentioned

causes

BIOPHYSICAL

synthesis

DEAE-cellulose

protein

1 ulose

M phosphate It

of

different.

the

AND

CAMP-independent

properties

quite

On

BIOCHEMICAL

and