OVEREXPRESSION AND PURIFICATION OF THE WILD-TYPE AND

MUTANT SIGMA FACTORS

2.1 Introduction

The sigma factor, a dissociable subunit of E.coli RNA polymerase,

encoded by the rp oD gene, is required for specific recognition of promoter

DNA. Being present in less than stoichiometric amounts relative to core RNA

polymerase (a.2~'~), regulation of the amount of this protein in the cell may

regulate the level of transcription (Burton et al., 1983) and changes in the

level of sigma synthesis may cause changes in relative promoter utilisation,

a means by which gene expression is controlled.

2.1.1 The Sigma operon

In E .coli, genes with similar functions are often found clustered on the

chromosome into organized transcription units called operons. Coordinate

control of gene expression is achieved by regulating the rate at which the

operon is utilized. The rpoD gene, coding for the sigma subunit (613 amino

acids), is within the operon that also has the structural genes, rp s U whose

gene product is the ribosomal protein S21 (71 amino acids), and the dna G

(581 amino acids) that codes for DNA primase (Fig.2.1) and located at -6 6'

on the E .coli chromosome. The three genes, whose regulatory features have

been identified, are present m the order rpsU, dnaG and rpoD and their

products are essential for translation, replication and transcription,

respectively. The direction of transcription of the rpsU, dnaG and rpoD

genes is clockwise relative to the E. coli genetic map. The sigma operon is

similar to the alpha and beta subunit operons in that the initial gene of the

operon codes for a ribosomal protein. But the sigma operon, unlike the

alpha and beta operons, codes for a protein, DNA primase, tha~ is essential

for replication. Burton et al., (1983) have also made a detailed analysis of

the regulation of the sigma operon wherein all three protein products play

a major role in the initial steps of the processes in which they are involved,

and therefore they are organized in a single operon (Wold and McMacken,

1982; Lupski et al., 1982a; 1983). That the genes are within the same

operon was confirmed (Burton et al., 1983) by Sl nuclease mapping of the

SIGMA OPERON

0~3kb •

~.3 lr.b

t,

RNA PROCESSING

J .. ----------------~2-~t~kb~------------~•mRNA

rpoD (Stgma)

Fig.2.1: Schematic structure and regulatory features of the· sigma operon:

Major operon promoters are designated as P and the promoter

activated by temperature upshift is denoted as PHs. Terminators

. are indicated as stem and loop structures (Burton et al., 1983).

in vitro transcripts. A 2.1 kb transcript coding for the sigma subunit begins

at the RNA processing site near the 3' end of the dnaG gene and extends to

the terminator downstream from rpoD (Fig. 2.1). During exponential

growth, the. steady state levels of proteins encoded by the sigma operon

genes have been estimated at 8000 copies of sigma (I wakura et al., 197 4) 50

copies of DNA primase (Rowen & Kornberg, 1978) and 50,000 copies of S21

(Kjeldgaard & Gausing, 1974).

In the past, studying the structure and functions of the sigma factor

was limited by the difficulty in obtaining sufficient amounts of protein

which is present in low amounts in the cell. The purification of sigma factor

was then controlled by ·the purity of the starting material or RNA

polymerase and then separating the core RNA polymerase from the sigma

factor by the method of Burgess & Travers (1971), and Lowe et al., (1979).

· Core RNA polymerase purified in the laboratory was used to reconstitute

holoenzyme containing the wild-type and mutant sigma factors which were

used for in vitro transcription experiments.

2 .1.2 Features of the plasmid pGEMD

The plasmid pGEMD (Fig.2.2), which overproduces the sigma protein,

was obtained from the National Institute of Genetics, Mishima, Japan. The

rpoD gene in this plasmid is selectively and actively transcribed by T7 RNA

polymerase in an expression system, BL21(A.DE3) (Studier and Moffatt, 1986;

Studier et. al., 1990), and produces large amounts of the protein on

induction with IPTG. The original construct of the plasmid pGEMD, was

made by treating the expression vector pGEMEX-1 (Promega) with Xbal and

self ligated to delete the T7 gene10 region. The pOEM ~ Xbal was used to

clone the 2.1kb Saci-Eco01091 fragment containing the entire rpoD gene.

The 2.1kb fragment obtained from the plasmid pYN3 was treated with

Klenow and then ligated with Hindlll linkers before inserting into the

Hindlll site of pOEM ~ Xbal.

2.1.3 Criteria for the purification of the sigma protein

The purification of the sigma factor relies on three properties of this

protein. (a) Rapid and complete renaturation after treatment with

guanidinium hydrochloride, a denaturant used to extract the protein from

30

r Amp

Fig. 2.2: Circular map

The rpo D

under the

indicates the

Hind Ill

pGEMD (5212 bp)

Hind Ill

' T7 promoter

of the overexpression plasmid pGEMD:

gene is represented by the thick darkened line and is

T7 promoter (open rectangle). The thick arrow

direction of transcription of the rpoD gene.

the cell debris, (b) strong binding to DEAE cellulose as it is acidic in nature

(pi 5.2) and (c) high stability and its relatively large size (Gribskov &

Burgess 1983). The host strain for the plasmid pGEMD is BL2l(DE3).

Expression of the wild-type and mutant sigma factors is driven by the T7

promoter which is selectively transcribed by T7 RNA polymerase synthesized

by the host strain BL21 (DE3), which has a single copy of the structural gene

for T7 RNA polymerase integrated into its chromosome, under the control of

the inducible lac UV5 promoter (Studier et al., 1990).

2 .1. 4 Inclusion bodies

In E. coli, overexpression of cloned genes may result in the formation

of intracellular proteinaceous granules that are visible under a light

microscope. The overexpressed protein is often found to be an insoluble

aggregate and is sedimentable by low speed centrifugation and located as

inclusion bodies in E.coli (Kane & Hartley, 1988). Various reports exist

indicating no relationship between the formation of inclusion bodies and

the promoter used to regulate the expression of the protein (Schumacher et.

aL, 1986, Posfai et al., 1986). Several parameters related to the host, the

growth conditions and the properties of the protein expressed could affect

the formation of inclusion bodies and is reviewed by Kane and Hartley

(1988).

The availability of large amounts of protein produced by using the T7

expressiOn system has facilitated the purification and characterization of

this transcription factor in order to study in more detail, its structure and

domain-wise functions.

2.2 Materials

2.2.1 Chemicals

Guanidinium hydrochloride was from Serva, Tris base, OTT, PMSF,

lysozyme, bisacrylamide, trypsin, sodium deoxycholate, chymotrypsin and

IPTG were from Sigma. Ultragel AcA44 was from LKB, DEAE cellulose was

from Whatman. Glycerol, Tris Buffer, EDTA, MgCl2, NaCl were of AnalaR

grade. Bacto tryptone, Yeast extract and Agar were from either Difco or

Himedia. 3H-UTP was from Amersham. NTP's were from Pharmacia and were

3 1

.. checked for purity by thin layer chromatography over polyethyleneimine

plates (Schlief & Wensik, 1981). Electrophoresis grade acrylamide was from

Serva or Jannsen. Low molecular weight protein markers were from Sigma

and glycine from Qualigens. All other chemicals and reagents were of

highest purity.

2.2.2 Bacterial strains and media

BL21(A.DE3) was streaked on LB agar plate and single colonies,

obtained after two passages, were used to inoculate liquid cultures. DH5a

and BL21 (DE3) cells harbouring the plasmid pGEMD or its derivatives as

described in materials and methods of chapter 3, were grown in sterile LB

medium containing 10 g tryptone, 5 g of Yeast extract and 10 g of sodium

chloride in 1 litre of single distilled water in the presence of ampicillin at a

final concentration of 100J..Lg/ml for 1.5% agar plates and 50J..Lg/ml for the

liquid culture.

2.2.3 Buffers and solutions

Lysis buffer - 1x contains 50mM Tris.HCl pH (8.0), 5%(v/v) glycerol, 2mM

EDTA, 0.1mM DTT, lmM ~-mercaptoethanol, 0.233M NaCl, 130J..Lg/ml

lysozyme and 0.1mM PMSF.

TGS buffer- 25mM Tris.HCI (pH 8.0), 192mM glycine and 0.1% SDS.

TGED buffer- This is the buffer used during the entire protein purification

and a 4x stock was prepared. A lx composition containslOmM Tris.HCl (pH

8.0), 5%(v/v) glycerol, O.lmM EDTA and O.lmM DTT.

Storage buffer: All purified preparations of sigma and core RNA

polymerase were stored at -20°C in lx TGED (pH 8.0) + 0.5M NaCl with 50%

glycerol.

Sample loading buffer for . SDS gels: A 2x stock containing 125mM

Tris.HCI (pH 6.8), 4% SDS, 10% ~-mercaptoethanol, 20% glycerol and 0.04%

bromophenol blue.

, TE buffer: lOmM Tris.HCl (pH8.0), 1mM EDTA.

32

2.3 Methods

2.3.1 Growth of cr 7 0 overproducing cells

BL21 (DE3) cells harbouring the plasmid pGEMD or the mutant

plasmids pVM-1, pVM-2, and pVM-3, were grown in LB medium containing

ampicillin at a concentration of 50f.lg/ml and incubated overnight at 37°C.

2 ml of the overnight culture was used as a starter culture to inoculate fresh

200 ml of LB + ampicillin medium and when cells reached the mid-log

phase of 0.6-0.7 OD(A600), IPTG was added to· a final concentration of

lmM and the incubation at 37oc was continued for 180 min. The cells were

then harvested and stored at -20°C till further use.

2.3.2 Analysis of the overexpressed sigma factor by

SDS-PAGE

Samples of (1 ml) harvested cells were centrifuged and the cell pellet

resuspended in 500f.ll of 50mM Tris.HCl (pH 8.0). The cell suspension was

repelleted again and resuspended in 150f.ll of sterile water by vigorous

vortexing to lyse the cells. An equal volume of sample buffer was added and

mixed well by vortexing and then heated in a boiling water bath for 3'. Cells

equivalent of 0.6 O.D. were analyzed on a 10% SDS page in a Hoefer system

at 40 milliamps constant . current. The discontinuous buffer system of

Laemmli ( 1970) was used and the gel stained with 0.2% coomassie brilliant

blue in 10% acetic acid, 45% methanol and 45% distilled water and

subsequently destained with the same solution without coomassie blue, till

the bands were visualized on a clear background.

2.3.3 Purification of sigma factor

After analysis of the crude cell extracts, on SDS gel, for the presence of

overexpressed sigma factor, the larger aliquot of cells pelleted from a 200 ml

culture was used for further purification using the method of Gribskov and

Burgess(1983), which was scaled down. A few modifications m the

chromatography steps was made wherein Ultragel AcA44 was substituted for

the Sephacryl S-200 in the gel filtration step.

33

2.3.3.1 Lysis of cells

Cells (0.6g wet weight) obtained from a 200ml culture were

resuspended in 2.5 ml of lysis buffer, in a 35ml polypropylene tube and

incubated for 30' on ice. Sodium deoxycholate was then added to a final

concentration of 0.05% to the suspension and further incubated on ice for

another 15'. The viscous solution was then transferred to a 50ml

polypropylene beaker and sonicated thrice with a Branson sonifier for 30

sees. at a time, at maximum setting. The sonicated mixture was then

diluted to 5 ml with TGED + 0.2 M NaCl and spun at 8000g (8500rpm) in a

SS-34 rotor precooled to 4oc. Earlier studies on the purification of the sigma

factor have shown a large portion of the sigma factor remains associated

with the cell debris. The supernatant was discarded and 2.5 ml of TGED +

0.2M NaCl was added and the pellet resuspended by gentle pipetting with a

pasteur pipette. This was then centrifuged at 8000g and the pellet stored on

ice. The cell debris at this step was grey brown in colour and most of the

overexpresed protein, present as inclusion bodies was in the cell debris.

2.3.3.2 GuHCI denaturation

The large amount of protein associated with the cell debris was

extracted with 0.8 ml of TGED + 6M GuHCl in a 2 ml hand-held

homogenizer with a

centrifuged at 12,000g

teflon pestle and the coloured suspension was

for 30', 4°C to remove the insoluble material. This

volume of GuHCl was necessary to prevent reaggregation of sigma in the

subsequent steps of dilution.

2.3.3.3 Renaturation by dilution

The supernatant after centrifugation, was transferred to a beaker, and

diluted in steps to a volume of 48 ml with a 10' interval between each

addition of 5 ml, 10 ml, 15 ml and 18 ml of TGED. The mixing during the

dilution steps was done in cold with the help of a magnetic stirrer; the bead

kept at slow stirring. A turbid solution formed during the dilution step was

pelleted by centrifugation at 8000g. The supernatant was further diluted to

96 ml and stirred slowly for 4-5 hrs, on a gyrotary shaker at 4oc with 0.5

gm of Whatman DE-52 resin.

34

2.3.3.4 DE-52 chromatography

The resin was allowed to settle and the slurry was packed in a 5 ml

disposable syringe of diameter 1.5 ems. The column was equilibrated with

TGED + 0.18 M NaCl at 24 ml/hr until the A280 was 0.05. The protein was

then eluted with TGED + 0.28 M NaCl and the washing continued till the

eluate showed an A280 of 0.05. The peak fractions (Fig. 2.3), having an

A28Q/A260 of 1.5-1.7, was then concentrated by the addition of 0.42 g/ml of

solid ammonium sulphate (to 60% saturation) and stirred in cold, on a

magnetic stirrer for half an hour. Later, the ammomum sulphate

suspension was pelleted by centrifugation for 30' at 4200g and the pellet

resuspended m 880J.Ll of TGED + 0.5 M NaCl.

2.3.3.5 Ultragel AcA44 chromatography

The ammomum sulphate pellet obtained from the previous step, was

resuspended in TGED + 0.5 M NaCl, and applied to Ultragel AcA44 gel

filtration column (10 ml bed volume) equilibrated with TGED + 0.5 M NaCl

and eluted with· the same buffer. The flow rate was set at 30 ml/hr and 1 ml

fractions were collected. The peak fractions (Fig. 2.4), were pooled and the

purity determined by analysis on a 10% SDS page. The approximate yield

was 3-5 mg for the wild-type sigma factor and the average recovery of the

pure fraction was approximately 1 mg from a 100 ml culture. Pure fractions

were pooled and extensively dialysed against TGED + 0.5 M N aCl with 50%

glycerol and stored at -200C in small aliquots.

2.3.4 Purity and yield

Protein samples from various stages of purification were heat.

denatured with 2x sample buffer and analyzed on a 10% SDS PAGE; 10%

separating gel (pH 8.8) and 5% stacking gel (pH 6.8) (Fig.2.5). The buffer

used for electrophoresis was TGS (pH 8.3) and the samples were

electrophoresed till the bromophenol blue reached the bottom of the gel.

Sigma factor of highest electrophoretic purity (-98%) was used to

characterize the activity and other biochemical functions. On storage, a

minor contaminating band of -66 kDa appears, depending on the amount

of protein loaded. The coomassie blue stained gels were dried and scanned

35

Fig.2.3:

0 <X> C\J

<t

0·5

0·4

0·3

0·1

16 20

FRACTION NUMBER

Chromatography of sigma factor on DE-52 column:

One ml fractions were collected and their A280/260 values checked

and peak fractions pooled.

Fig.2.4:

0 00 C\J

<t

0·3

0·2

0·1

2 5 8 10

FRACTION NUMBER

Gel filtration chromatography of "DE-52 purified sigma

factor"on Ultragel AcA44:

One ml fractions were collected, checked for A280/260 values and

peak fractions pooled.

2 3 4 5 6 7 8 9 10

cr - .... - --

-- ... -- -

Fig.2.5: Purification stages of the wild-type sigma protein 10% SDS polyacrylamide gel analysis. Lane 1 Core RNA polymerase; . Lane 2 GuHCl extract spill over from Lane 3; Lane 3 GuHCl homogenized extract; Lane 4 GuHCl extract of pellet; Lane 5 Supernatant after dilution steps; Lane 6 Pellet after precipitation of diluted extract; Lane 7 Crude cell lysate; Lane 8 Blank; Lane 9 Supernatant after TOED + 0.2M N aCl wash; Lane 10 Peak fraction of DE-52,50 )ll (10 )lg)

usmg a densitometric scanner to determine the amount of protein

expressed.

2.3.5 Quantitation

Protein concentration was estimated by the method of Lowry et al.,

(1951) and also spectrophotometrically at 280nm; a region of absorption of

largely the tyrosines and tryptophans. The value of £ can be calculated for

proteins by adding up the contributions of the constituent aromatic amino

acids. (Wetlaufer,1962; Scopes, 1974). The absorption of proteins in the 230-

300nm range is determined by the aromatic side chains of tyrosine,

tryptophan, and phenylalanine. The molar absorption of phenylalanine is

smaller by an order of magnitude than tyrosine or tryptophan, the latter I

being the most strongly absorbing amino acid. Therefore, the UV absorption

spectrum is largely the contributions of tyrosine and tryptophan, which

depend on the nature of the molecular environment of the two

chromophores (Schmid, 1989). Purified sigma factor has an extinction

coefficient of A% 280 = 8.4, values reported by Lowe et al.,(1979) from which

the concentration was determined. In a good preparation, the A280/260

ratio was approximately 1. 7 which indicates that there is no nucleic acid

contamination. In some batches, however, the A280/A 260 ratio was greater

than 1.5; 1.e. 0.5% nucleic acids and the concentration of the protein was

corrected using the formula:

Protein concentration (mg/ml) = A280 * F, where F is the correction factor

(Boyer, 1986).

2.3.6 Molecular weight determination of sigma factor

The molecular weight of proteins can be determined by either SDS

-PAGE or by gel filtration. The molecular weight of sigma factor was

determined by running a 10% SDS page using the standard low molecular

weight markers to generate a calibration curve. The procedure of Weber and

Osborn (1969) was adapted for the measurement of the relative mobility

(Rf). The molecular weight of the wild-type and mutant sigma factors was

deduced from the known Rf value by interpolation from the graph. The

estimation of the molecular weight by gel filtration was also performed

using the HPLC system, Waters 1-125 column (2000-80,000 range) in a buffer

36

containing 50mM KCl, Tris. HCl (pH 8.0), 10mM MgCl2 and 5% glycerol and

at a flow rate of 1 ml/ min.

2.3.7 Purification of core RNA polymerase

2.3.7.1 Materials

Phosphocellulose (Whatman P11), RNA polymerase was prepared by

the method of Burgess and Jendrisak, (1975) and further improved

according to Kumar and Chatterji, (1988). Buffers and solutions were as

described in the earlier section and all other chemicals were of highest

pori ty.

Phosphocellulose was stirred with 5 volumes of 0.5M N aOH for 20' and

filtered on a sintered-glass funnel 02 and rinsed with MilliQ water until the

pH of the rinse was about (8.0). The cake obtained after suction of the rinse

using a vacuum line, was stirred with 5 volumes of 0.5M HCl, 20' and once

again passed through a sintered funnel until the pH of the rinse was (6.0).

The phosphocellulose was then resuspended in 2 volumes of 50 mM

Tris.HCl, 50 mM KCl and adjusted to pH(8.0) with 6N KOH. The titration

with KOH was done over a length of time of -6 hours and then equilibrated

with a buffer containing 50mM Tris. HCl (pH 8.0) (250C), 0.1 mM EDTA, 0.1

mM DTT and 5% glycerol.

2.3.7.2 Method

E.coli RNA polymerase purified in the laboratory was the starting

material for the purification of core RNA polymerase. 2 mg of holoenzyme

was checked for its purity and then dialyzed overnight against 500 ml of

TOED + 50mM KCI. The. dialyzed sample was then applied onto a

phosphocellulose column, equilibrated with TOED + 50 mM KCl, and set at

a flow rate of 24 ml/hr. A single step slow elution with TOED + 0.5M KCl was

done and the fractions were collected and checked for A280· The core RNA

polymerase obtained was again reapplied to the phosphocellulose column

and the procedure of washing and elution repeated (Fig. 2.6). Peak fractions

were pooled and dialyzed extensively against TOED + 50% glycerol + 0.1 M

NaCl and the activity checked using LlD111T7A1 as the template (Nierman

and Chamberlin, 1979).

37

Fig.2.6:

0 co N

<!

0·5

0·4

0·3

0·2

0·1

8 12

FRACTION NUMBER

Chromatography of RNA polymerase on phosph,ocellulose column: Purification of core RNA polymerase.

The purification of core RNA polymerase relies on the principle that

sigma factor does not bind to phosphocellulose and is largely in the flow

through, while core RNA polymerase binds to the column and was seen to

elute at -0.35M KCI.

2.3.8 Reconstitution of bolo RNA polymerases

Equimolar amounts of pur~fied sigma factor and core RNA

polymerases were taken and dilutions, where necessary, were done with lx

TGD [10mM tris.HCl (pH 8.0), 5% glycerol and 0.1mM DTT]. These were

mixed properly, and incubated for 15' at 370C before use.

2.3.9 In vitro t r a n s c r i p ti o n

Sigma activity was measured by its ability to stimulate transcription

ofdD111T7A1 (Nierman and Chamberlin, 1979) containing a single, strong

A1 promoter. as the template. The assay mixture in 100J.Ll was 40mM tris.HCl (pH8.0), 200mM NaCl, lOmM MgCl2, lmM EDTA, 200J.LM each of CTP, GTP,

and ATP, 50J.LM UTP, lJ.LCi of 3[H]UTP, 3.5 J.Lg of dD111T7Al and 1mM DTT

(Lowe et. al., 1979). The template was preincubated in the presence of the

assay mix containing the substrates and the respective reconstituted

enzymes at a 1:4 molar ratio (DNA:holoenzyme) and incubated for a period

of 20' and subsequently spotted on DE-81 filters previously soaked with

EDT A. On drying, the filters were washed thrice with 5% sodium phosphate

dibasic, to remove all unbound products of the reaction, twice with water

and a final rinse with clear, crude ethanol as described by Somers & Pearson

(1975), to remove all traces of water. The dried filters were then placed in

scintillation vials containing the toluene based scintillation fluid and

counted (Dyan et al., 1977).

2 . 4 Results & Discussion

2.4.1 Overexpression of the sigma subunit

Fig 2.2 shows the plasmid map of pGEMD harbouring the rpoD gene.

The total cell protein after induction with IPTG, was checked on a 10% SDS

page. As seen in (Fig.2.7a) and the densitometric scan trace of lanes 1 and ·

2 3 4 5 6

Fig·. 2. . / (z~ : ()v erex ,. .-:-~ s w n of ;-;t ,l.~ t·. w-r; BL1l r~ E:3 ) .cdJ. ~ c .. il,l~n1 F. tk .~h ·n i J. p. r ~ MU was grr.n.r:J 1.0 rei.d-I.og ~ha~ '"' · t. rt th t~ m u~.~rl wi 1~ LPT . J .<me. J: Ri lfi .. pci.:rmeras"' f 'L'.g I i "l. i. ~ J <, It(, ~r ; r .ane 1· 6~ t j n!'c· ,,muse of r,{,L ~ i:1 r ho X'.il!r, t1 .c l as mi ci pn.Givi f} ':. \~~;f .ssir g the ,.1\· o ~YP~ ;; ig:rmt ht·t . ']'ow l c<~1 -··J' 'r., .~ er :.:1e (2 _:_· •) 1:; -..q 1'vc cu t to 0. · O.D. Ln 'tC, 1., :~ ::. ; b :: .. e 3, 1 · _s.i.:!.H, 1 ut:: <-}, 4 min; l twe 5, 50 m r!; r.a.::-~c ·, -o min · .. L~r . :r dac lon with IPTG .

6 of the same figure in F" g 2. 7b, (i) and (ii) , sigma constitutes 33% of the

total cell protein. Even in the case of the mutant s ig~a factors W433F,

W434G and W433G, (Fig. 2.8 a & b), there was a significant increase in the amount of protein expressed and in the case of cr 7 0 _ W 434G , the

overexpressed protein migrated as a 70 kDa protei n. There was no

significant increase in the expression levels even 180 minutes after IPTG

induction, and the cells were harvested after this time period. The mutant sigma factors cr 7 0 _ W 433G and cr 7 0 _ W 434G, in uninduced cultures, grew

much slower (Fig 2.9). However the yield of the protein was almost

comparable to that of the wild-type s'gma factor.

In the case of the plasmid pVM-4 ( W433/434G), which is a double

glycine mutant, there was no expression of the protein owing to the toxicity

of the gene (not shown). This may also be due to plasmid instability, which

arises when the gene product cloned in the plasmid · s toxic to the host cell

(Studier et al., 1990). Methods to reduce basal level expression of the double

glycine mutant, in more tightly repres ·ed systems, were attempted. Also the

presence of pLysS or pLysE, which 'ncreases the tolerance of BL21(DE3) for

toxic target plasmids did not help in overexpression of the protein. In yet

another strategy to ove,.express the double glycine mutant sigma factor,

infection of HMS 1'1 4 cells, transformed with this plasmid, with

bacteriophage CE6, .~ A. derivafve carrying the gene for T7 RNA polymerase

was attempted bu · with no success.

2.4 .2 Activity of cr 7 0 and its mutants cr 7 0 • W 4 3 3 F , cr 7 0. W 433G & cr 7 0. W 3 4 G

The yield of core RNA polymerase was about 1.6 mg and used for the

reconstitution of holoenzyme containing the wild-type and mutant sigma factors which are hereafter called cr 70 _ W433F, cr 70 _ W434G and cr 70 _ W 43 3G.

The native sigma factor will be referred to as cr 70wild-type. The purity of

core RNA polymerase was checked by electrophoresis on 10% SDS gels. Fig.

2.10 shows the band pattern of the core RNA polymerase on such a gel.

The reconstituted enzymes were checked for their ability to transcribe

promoter specific templates. It can be seen from the bar diagram (Fig.2.11),

that there is maximal stimulation of core RNA polymerase activity (-12 - 14

fold) in the presence of the wild-type sigma factor as well as cr 70_W433F i.e.

39

1

3

Fig.2.7(b): Densitometer scan trace of RNA polymerase and sigma factor: Lanel and Lane 6 of Fig. 2.7(a) were scanned. Numbers over the main peaks in (i) correspond to subunits ~ ·~, peak 1; cr, peak 2; a., peak 3 of RNA polymerase, and (ii) peak 3 corresponds to 33% (sigma) of total cell protein.

( i )

3

(i i)

12

a

1 2 3 4

J3'f3- --· cr- cr7Q. W433F b

1 2 3 4 5

<t-70 -a -W433G 70

- 0'" -W434G

Fig. 2.8(a) Overexpression of cr 70_ W 433F: BL21(DE3) cells containing the plasmid pVM-1 was grown to mid­log phase and then induced with IPTG. Lane 1, 10 J..Lg of RNA polymerase; lane 2, 0' ; lane 3, 60' ; lane 4, 120' after induction with IPTG.

(b) Overexpression of cr70_W434G & cr70_W433G: BL21(DE3) cells containing the plasmid pVM-2 or pVM-3 was grown to mid-log phase and then induced with IPTG. Lane 1, 0', lanes 4, 60'; lanes 5, 120' after induction with IPTG. Lanes 2 and 3 are overexpression of cr 70_W434G, 60' and 120' after induction with IPTG and loaded to compare mobility differences with the major protein band in lanes 4 and 5.

5·0-------------------------------.

4·0

E c 0 3·0 0 <.D

0 0

2·0

I ·0

Fig. 2.9:

2 3 4 5 6

Time , hr.

Growth curve of BL21(DE3): cells contammg the wild-type and mutant sigma factors .

7

Mutant sigma factors FW ( o ), WG ( • ) and GW ( c ) correspond to a 70_W433F, a /O_W434G and a 70_ W 43 30 respectively. The wild-type sigma factor is referred to as WW (~.

1 2

_ cr70

-cc

Fig. 2.10: 10% SDS-PAGE profile of core RNA polymerase: lane 1, 4 11g of purified sigma factor; lane 2, 15 11g of purified core RNA polymerase.

60

50

(') 40

0 ..... )(

E 30

c.. (.1

20

10

0 core enzyme

WN FW W3 GN

Fig. 2.11: DE-81 bound counts of the wild-type and mutant cr factors: Bar diagram to show relative activity at .1.DIIIT7 Al template, when reconstituted with core RNA polymerase.

when the tryptophan at position 433 was substituted with phenylalanine.

This was expected as W and F are like amino acids and this replacement

should not result in any change in its structure or its activity. In contrast,

cr 70_W433G and cr 70_W434G show almost basal level activity similar to that

of core RNA polymerase. Also, both cr 70_W434G and cr 70_W433G bind core

RNA polymerase and the abortive initiation assay at T7 A1 templates suggest

that these mutants are as efficient as the wild-type sigma factor at

supercoiled templates and initiate with lesser efficiency at linear templates.

However, total transcription using cr70_W434G and cr70_W433G reconstituted

enzymes at linear DNA fragments showed poor transcriptional activity and

has been discussed in more detail in chapter 4.

2.4.3 Physicochemical properties

2.4.3.1 Molecular weight

The molecular weight of 70,263 daltons has been calculated for the

613 amino acid polypeptide (Burton et al., 1983). This molecular weight is

20% less than the apparent molecular weight of 87,000 daltons of the wild­

type sigma factor, as determined by SDS polyacrylamide gels in a

discontinuous buffer system. The comparative mobility of the wild-type

and mutant sigma factors, with the low molecular weight markers, is shown in (Fig.2.12), as a linear plot of Rf vs log molecular weight. The anomalous

mobility of the wild-type sigma factor as deduced from the graph is 87 kDa,

reported before by Burgess. The molecular weight of the mutant cr70_W434G

on SDS gels is 70 kDa, which is the expected mobility of this 613 residue

protein. Anomalous electrophoretic mobilities have been reported for a

number of proteins analysed on SDS gels (Seeburg et al., 1984, Fasano et al.,

1984). Some proteins are known to contain a large amount of secondary

structure in the presence of SDS (Jirgensons, 1970; Jirgensons, 1982; Mattice

et al., 1976). The reason for the anomalous mobility on SDS gels is not clear

and could also be due to a differential binding of SDS monomers. In the

case of sigma factor, this .could be due to a stretch of negatively charged

aspartic acid residues in the dispensable region (130-374), of the sigma

polypeptide, which is not conserved in cr43 of B. sub til is . As seen from the

calibration curve (Fig. 2.12), the molecular weight of cr70_W434G is 70 kDa;

the actual, expected molecular weight. Gel filtration, by HPLC, shows

identical elution profiles of the wild-type and the mutant sigma factor cr 70_

40

I (3 (3- 155/150

Fig.2.12: Determination of the molecular weight of cr factor: Plot of Rf versus log MW (linear scale) for standard proteins ranging from Mr 67,000 to 14,000 (open circles) & W~ and a (closed circles). The molecular weight of cr · was obtained by interpolation.

W 4340 which elutes as a 70 kDa protein (Fig. 2.13). This indicates that the

wild-type and two of the mutants cr70_W433F and cr70-W433G which show

anomalous mobility on SDS gels, are not dimers.

2.4.3.2 Spectrophotometric properties

The ultraviolet absorption spectrum of purified sigma factor is shown

in Fig 2.14. The protein has a A280/260 ratio of 1.7 which indicates no

detectable contamination with nucleic acids. The extinction coefficient of

8.4 reported before by Lowe et al., (1979), was used to determine the

concentration of the protein and is in agreement with the values obtained

by Lowry's method where BSA was used to generate the standard curve.

41

E c 0 (X) (\J

+-0 Q) u c 0

..Q ~

0 Cl)

..Q

<t

........

3: (!)

3 3: 0 0 <t

"" ""b (/)

b £D

\ l~~ 11)(0<0

CDCD 1010

' ( I

j I I ) I I

! J I I I I I

j I

i J I

i \ I I I CD I

~·l CD

l . = . ~ .....

_____ ))/ I_ - ; ···-· ·-·-····---·r I I

Time (minutes)

Fig. 2.13: Gel filtration by HPLC of purified cr 70wild-type(WW), cr70_W434G(WG) and BSA. (bovine serum albumin).

I&J 0 z <t m 0:: 0 CJ)

m <(

260 280 WAVELENGTH

Fig. 2.14: Absorbance spectrum of purified sigma factor