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Repression of Leucyl-transfer ribonucleic acidsynthetase of escherichia coliHarold L. WashingtonAtlanta University

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ABSTRACT

BIOLOGY

WASHINGTON, HAROLD L, B.S., Clark College, 1968

Repression of Leucyl-Transfer Ribonucleic Acid Synthetase of

Sscherichia coli

Advisor: Dr. Luther S. Williams

Master of Science degree conferred June 1, 1970

Thesis dated June, 1970

This study was undertaken to determine whether the for

mation of leucyl-transfer ribonucleic acid synthetase (tRNA

synthetase) was "constitutive11 in nature or was subjected to

some control mechanism. The growth of wild type cultures in

minimal-glucose medium supplemented with excess leucine, as

compared to minimal-glucose medium, did not affect the

specific activity of the leucine synthetase. Using an auxo-

troph for leucine, a shift of this culture from a medium

containing excess leucine to restricting (growth rate)

amounts of leucine, resulted in a significant increase in the

specific activity of the leucyl-tRNA synthetase for the

first hour of incubation. This indicated a derepression in

the rate of formation of the enzyme. After the first hour of

incubation, there was a decline in the rate of synthesis and

some inactivation of this synthetase activity. Further

studies provide results that the addition of leucine to the

highly derepressed cultures caused repression of synthesis

and halted the inactivation of the leucyl-tRNA synthetase.

These results confirm previous findings indicating that the

rate of formation of arginyl- and histidyl-tRNA synthetase

was regulated, and suggest that the regulation of aminoacyl-

tRNA synthetase formation follows a repression-like

mechanism.

Master of Science Thesis

of

Harold L. Washington

Approved:

Major Professor

Thesis Committee Member

Thesis Committee Member / ,

Department Chairman yS .

Dean, School of Arts and

REPRESSION OF LEUCYL-TRAN5PER RIBONUCLEIC ACID

5YNTHETASE OF ESCHERICHIA COLI

A THESIS

SUBMITTED TO THE FACULTY OF ATLANTA UNIVERSITY

IN PARTIAL FULFILLMENT OF THE REQUIREMENTS

FOR THE DEGREE OF MASTER OF SCIENCE

BY

HAROLD L. WASHINGTON

DEPARTMENT OF BIOLOGY

ATLANTA, GEORGIA

JUNE 1970

TABL2 OF CONTENTS

Page

ACKNOWLEDGEMENTS vi

LIST OF TABLES vii

LIST OF FIGURES viii

Chapter

I. INTRODUCTION 1

II. REVIEW OF LITERATURE 3

III. MATERIALS AND METHODS 5

IV. EXPERIMENTAL RESULTS 9

V. DI9CUS3ION AND CONCLUSION 24

VI. SUMMARY • • 25

LITERATURE CITED 26

ACKNOWLEDGEMENTS

The author wishes to express his sincere appreciation to

Dr. Luther 3. Williams for his guidance during this research

and the preparation of this thesis. The author also wishes

to thank Dr, Roy Hunter, Jr. and Dr. Joe Key for their

suggestions on the writing of this thesis.

vx

LIST OF TABLES

Table Page

1. Effect of L-leucine limitation on specific

activity of leucyl-transfer ribonucleic acid

synthetase of strain AB1132 ... 11

2, Effect of leucine deprivation on the specific


synthetase. ••••••• ••••••••••••13

3# Effect of leucine starvation on the specific


synthetase •••••«••••••••••••• 16

vii

LIST OF FIGURES

Figure page

1. Effect of excess leucine on the specific activity

of leucyl-tRNA synthetase of strain AB1132, ... 10

2. Effect of leucine restriction on the specific

activity of leucyl-tRNA synthetase of strain

AS1132 • . • 14



A31132 for duplicate cultures* •••• 17


activity of leucyl-tRNA synthetase for duplicate

cultures of strain AB1132 at 2 minute intervals, • 18


activity of leucyl-tRNA synthetase for millipore

filtered cells of strain AB1132 .,20


activity of leucyl-tRNA synthetase after two

days of incubation, •••.,.,•••••••, 21

7. Comparison of percent charging of leucine to

leucyl-tRNA for a control and sodium periodate

treated sample , ••••••«•«••••••• 23

viii

CHAPTER I

INTRODUCTION

It has been a rather consistently asked question as to

whether aminoacyl-tRNA synthetase was "constitutive" in

nature or was subjected to some control mechanism. However,

since there are 20 activating enzymes in Escherichia coli,

the rate of formation for each enzyme is conceivably differ

ent in nature and magnitude.

Leucyl-tRNA synthetase is responsible for the attachment

of leucine to its specific tRNA. The amino acid, leucine,

is activated with ATP and the leucyl-tRNA synthetase to form

leucyl-AHP-enzyme complex. Once the amino acid becomes

activated, its specific synthetase enzyme, leucyl-tRNA

synthetase, attaches it to the terminal nucleotide on the

tRNA molecule for leucine.

Leucyl-tRNA synthetase formation could result from the

following conditions: (1) Synthesis of the synthetase could

be constitutive in nature. (2) The leucyl-tRNA synthetase is

inducible9 with its synthesis being induced by its sub

strates, which are leucine and its specific tRNA molecule.

(3) The formation of this enzyme could be regulated by end-

product repression.

This study of leucyl-tRNA synthetase was undertaken to

determine the controlling mechanism for its formation*

CHAPTER II

REVIEW OF LITERATURE

A number of studies has been made on aminoaeyl-tRNA

synthetase regulation in B. coli but it is not yet understood

in any depth. During the growth in any media, prototrophic

E. coli cells are able to maintain a nearly constant level of

the individual araino acid activating enzymes,

Nass and Neidhardt (1967} reported that most of the

evidence suggested that the synthesis of these enzymes is not

"constitutive". However, Boman, Roman, and Maas (1961} re

ported that the removal of arginine from a minimal-growth

medium has been reported not to change the differential rate

of synthesis of arginyl-tRNA syntheses in B. coli., although

the synthesis of arginine biosynthetic enzymes was acceler

ated several fold under these conditions. Ames and Gary

(1962) have concluded that the histidyl-tRNA synthetase in

Salmonella, typhinmrium is not controlled by the histidine

operon, and has the same differential rate of synthesis

regardless of the concentration of L-histidine.

Nass and Neidhardt (1967) were able to provide evidence

that the synthesis of phenylalanyl- and isoleucyl-tRNA syn

thetase was not "constitutive" but is regulated by an amino

4

acid mediated repression. Restriction of either of the

phenylalanine or isoleucine supply to the cells resulted in a

derepression in their respective synthetase. From these re

sults, it was still impossible to conclude that restriction

of appropriate amino acid supply is sufficient to derepress

other synthetases. Restriction of histidine and leucine sup

ply failed to cause derepression of the histidyl and

leucyl-tRNA synthetases. It was suggested that either mul

tiple mechanisms exist for regulating the formation of these

enzymes, or amino acid restriction is not universally effec

tive in causing derepression of aminoacyl-tRNA synthetase

formation for some, as yet unknown, reasons,

MLlliams and Neidhardt (1969) have reported that many

of these enzymes are subjected to high rates of irreversible

inactivation during growth under amino acid restriction, es

pecially when the growth is intermittent. They have suggested

that all of the enzymes exhibit a repression-like mechanism

and can adjust their rate of synthesis over a 10 to 50 fold

range.

CHAPTER III

MATERIAL3 AND METHODS

Organi sm

The organism used in this study was a strain of the

bacterium, Escherichia coli. Strain AB1132 is a multiple

auxotroph for histidine, leucine, raethionine, proline, thre»

onine and thiamine. It was obtained from Dr. Luther S.

Williams. The organism was maintained on a glucose-tryptone-

yeast extract agar slant under refrigeration.

Medium and Method of Cultivation

In all experiments, the medium used was minimal-glucose

medium of Fraenkel and Neidhardt (1961) consisting of 0.046%

»7H2O, 1.34% Na2HPO4«7H2O, 0.0011% CaCl2, and 1.36%

at a pH of 6.5. This growth medium was supplemented

with 0.2% (NH4)2S^ and 0.2% glucose as nitrogen and carbon

sources respectively. Amino acids added to the medium were

of the L-form. Leucine was added to overnight cultures at

a final concentration of 50 Ag/ml and/or ISQ^g/nal. Ml

other amino acid supplements were supplied at a final

concentration of 50y^g/ml.

Batch cultures were grown aerobically in Erlenmeyer

6

flasks on a rotary action shaker (American Optical) at 37 C.

In all cases, cells were grown, at least, overnight in the

same medium to be used at the start of the experiment. At

the beginning of each experiment, the overnight cultures were

resuspended in minimal medium at an optical density (0»D«

at 0.2 to 0.4.

Measurement of Growth

The growth of all cultures was measured with a Turner

330 spectrophotometer by the increase in optical density at

420 mu(light path = 1 cm). The growth rate constant, k,

was determined for exponentially growing cultures using the

relationship:

k = In2

mass doubling time in hr

for batch cultures.

Protein Determination

Protein concentration of cell free extracts was deter

mined by the method of Lowry, Rosebrough, Farr and Randall

(1951). Standard protein concentration was determined by

using bovine serum albumin where the concentration ranged

from 20-100 /ig/ml.

Preparation of Cell Free Extract

Cell free extract was prepared by sonic treatment with a

Branson Sonifier Cell Disrupter (Branson Instruments, Inc.}

for 1 min at a setting of #3 followed by removal of cell de

bris by centrifugation at 10,000 rpm for 10 min.

Enzyme Assay

J~14~lAttachment of L- L, CJ atnino acid__tp_tRNA (attachment assay 1.

This assay was similar to that of Williams and Neidhardt

(1969). The reaction mixture, at a final pH of 7.3, in a

total volume of 0.5 ml, contained 0.1 ml of Tris-HCl buffer,

pH 7.3, 0.1 ml ATP, pH 6.8 at 0.02 M, 0el ml of tRNA, 0.1 ml

fl4 1of L L CJ leucine and 0,1 ml of enzyme extract. The com

plete reaction mixture was incubated for 15 min at 37 C, The

reaction was terminated by adding 3 ml of 5% trichloroacetic

(TCA) then chilled for at least 10 min. Suspension was then

filtered through a membrane filter (Whatman 2.1 cm GF/A).

The precipitated RNA was washed on the filter with 10 ml of

5% TCA followed by 5 ml of 67% ethanol. The washed filters

were dried by air and counted in a Packard Tri-Carb liquid

scintillation counter and/or thin-end window Nuclear-

Chicago gas flow counter.

8

Specific activity was expressed as units of enzymes /rag

protein.

Assay for percentage charged tRNA.

The cells were grown overnight in minimal-glucose medium

supplemented with 1— Cj leucine 150/4g/ml. At the begin

ning of the experiment, the cells were sub-cultured in fresh

minimal glucose medium with no leucine. The cells were

grown in this medium and 50 ml samples were taken at 6 and

12 min intervals. The samples were sonicated and cell de

bris removed by centrifugation. The tRNA was isolated by

phenol extraction and precipitated with cold ethanol, and then

redissolved in 0.1M sodium acetate buffer at a pH of 4,5.

The redissolved tRNA for 6 and 12 min samples, along with

an excess leucine sample, was divided into two samples of

equal volume, one of which was treated with 0.1M sodium per-

iodate. The second sample was used as a control. Both

samples were assayed for leucyl-tRNA synthetase activity;

however, the tRNA used was that obtained for treated and con

trol samples. Radioactivity of the samples was determined as

a measure of the percentage charging of leucine to its tRNA

of a control sample as compared to that of the periodate

treated sample.

CHAPTER IV

EXPERIMENTAL RESULTS

This study was made to determine the effect of leucine

on the specific activity of leucyl-tRNA synthetase and deter

mine whether or not the formation of this enzyme was

"constitutive" in nature or subjected to a control mechanism

in the cell. The data collected in this study indicated that

restricted concentration of leucine had a significant effect

on the specific activity of the leucyl-tRNA synthetase. This

is shown graphically in Fig* 1, in which the culture was

initially grown in medium containing leucine (30,/tg/ml) and

then shifted to a 50 Ag concentration of leucine, as a sup

plement to minimal medium. As shown in Table 1, the specific

activity of leucyl-tRNA synthetase decreased for the first

3 hrs of incubation, but upon the addition of excess leucine

(50/<^g/ral)9 the specific activity began to increase. These

data indicated that inactivation of the enzyme occurred in

limiting leucine culture and the addition of excess leucine

halted this inactivation process.

The leucine auxotroph strain AB1132 was grown in batch

culture (minimal medium containing leucine 50^g/ml) for

Fig. 1. Effect of excess leucine on the specific

activity of leucyl-tRNA synthetase of

strain AB1132. Cells were grown in

limited leucine concentration at the

indicated time. Appropriate samples were

taken at hour intervals and the activity

of leucyl-tRNA synthetase was determined.

Specific activity is expressed as^moles

of leucine attached to tRNA/hr/mg protein,

SPECIFIC

ACTIVITY

ot\>

Ui

<J\

O)

-4

CD

<O

I1

I—

c

-n

ro-

O

ai.

o

rn

i

m•

m o

11

Table 1. Effect of L-leucine limitation on specific activity

of leucyl-transfer ribonucleic acid synthetase of

strain AB1132.

Sample (hr|

1

2

3

4

5

Condition

Leucine

limited

it

ti

Excess

leucine

u

CPHa

2044

588

424

1536

1598

CPMb

511

147

106

384

397

Protein (rag)

0.0025mg

0.0015

0.0021

0.0038

0.0038

S.A?

0.8

0.39

0.201

0.404

0.421

CPU refers to counts per hour of L Cj leucine attached totRNA.

CPM refers to counts per min of L14cJ leucine attached totRNA.

c

S.A. refers to specific activity as expressed as moles of

leucine attached to tRNA per hour per mg of protein.

12

several generations. Upon transferring the cells to leucine-

free medium, appropriate samples were taken at specific

intervals and assayed for leucyl-tRMA synthetase activity.

As shown in Table 2, synthetase activity increased from

1.13 units/mg protein during growth in restricted culture to

2.74 units/mg protein for the first hr of incubation. However

as samples were taken for the remainder of the 5 hr of the

incubation period, the specific activity decreased to 0,77

units/mg protein. This shift from a leucine culture to a no

leucine culture caused the activity of the leucyl-tRNA syn

thetase to be inactivated. The rate of formation of the

leucyl-tRNA synthetase during derepressive growth conditions

and inactivation of activity is shown in Fig. 2.

The significant observation made in this study of the

auxotroph was the fact that leucine restriction resulted in

an inactivation of formation of leucyl-tRNA synthetase

activity. To ascertain the state of leucyl-tRNA synthetase

during each hour of incubation, it was necessary to perform

similar studies using this leucine auxotroph.

The strain was grown in batch (minimal medium containing

leucine, 150Ag/ml) culture for several generations. Upon

13

Table 2, Effect of leucine deprivation on the specific


synthetase.

Sample (hr)

0

1

2

3

4

5

Condition

Excess

leucine

No

leucine

it

u

it

ii

CPHa

1348

1918

1466

1548

334

538

CPMb

337

479

366

389

83

134

Protein (mg)

0.012

0.0007

0.0009

0.0011

0.001

0.0007

S.A?

1.13

2.74

1.63

1,36

0.334

0.77

CPH refers to counts per hour of

tRNA.

CPM refers to counts per rain of

tRNA.

C14 1, Cj leucine attached to

Cjleucine attached to

"S.A. refers to specific activity as expressed as/fmoles of

leucine attached to tRNA per hour rag of protein.

Fig. 2. Effect of leucine restriction on the specific


AB1132. Cells were grown in excess leucine

overnight and shifted to a no leucine

culture. Appropriate samples were taken at

the indicated time and the activity of

leucyl-tRNA synthetase was determined. Spe

cific activity is expressed as .Amoles of

leucine attached to tRNA/hr/mg protein.

SPECIFIC

ACTIVITY

IQ

ro

ro

ro

to

OCO

o

—I

m oro-

| w >CO

.

—i

en

%

o>

to

ro

en

oo

-*

30X3i■

i

CO

^S

CO

^/^

LEUCIf/

\ \

4^

-vjO

■i

i LEUCIm

15

transferring the cells to leucine-free medium, as duplicate

cultures, appropriate samples were taken at 30 tnin intervals

and assayed for leucyl-tRNA synthetase activity. In Table 3,

it can be seen that the specific activity of leucyl-tRNA syn

thetase increased from 0.214 to 2.001 units/mg protein for

the first hr of incubation, indicating a derepression of the

rate of formation of this enzyme. After the first hr of in

cubation, there was a decline in the rate of synthesis and

considerable inactivation of this synthetase activity (from

2.001 to 0.313). This rate of formation and concomitant

inactivation of activity of the leucyl-tRNA synthetase under

these growth conditions is shown in Fig. 3.

The derepression period shown in Fig. 3 was examined by

growing the cells in batch cultures supplemented with 150Ag

of L-leucine and transferred to a no leucine culture medium

and removing samples at 2 min intervals for a 15 min incuba

tion period. There was an increase in the rate of derepres

sion of formation of the leucyl-tRNA synthetase at a steady

rate (Fig. 4).

The procedure in this experiment was the same as des

cribed earlier, except that in the shift from excess leucine

16

Table 3. Effect of leucine starvation on the specific


synthetase.

Sample Condition CPHe CPM Protein (rag) S.A.*

3

4

5

6

8

9

No

leucine

it

tt

tt

tt

tt

it

u

it

1178

4854

9346

2270

9346

1536

I486

2808

7792

7148

1454

1010

1066

2912

2256

2892

294

1213

2336

567

2336

334

371

702

1948

1787

363

252

266

728

564

723

0.0055

0.00515

0.00495

0.00625

0.00467

0.0049

0.00522

0.0052

0.00515

0.00467

0.0045

0.00432

0.00501

0.00503

0.00515

0.00522

0.214

0.942

0.772

0.363

2.001

0.313

0.301

0.54

1.513

1.528

0.323

0.233

0.212

0.578

0.438

0.554

3CPH refers to counts per hour of L14CJ leucine attached totRNA. r

Ij leucine attached toI34cjbCPM refers to counts per min of

tRNA.

CS.A. refers to specific activity as expressed as ^moles of

leucine attached to tRNA per hour mg of protein.

Represents a calculation for duplicate cultures.



AB1132 for duplicate cultures. Cells were

grown in excess leucin© and transferred to a

no leucine culture. Appropriate samples were

taken at 15 min intervals and the activity of

leucyl-tRNA synthetase (•-♦;4-4 was determined.

Specific activity is expressed as zuaoles of


SPECIFC

ACTIVITY

E 15

o 01-

o-

rs>

<p

Fig,

120 135»lOSI

ro

-I-

»f

?■?

Fig. 4, Effect of leucine restriction on the specific

activity of leucyl-tRNA synthetase for dupli

cate cultures of strain AB1132 at 2 min

intervals. Cells were grown in excess leucine

and transferred to a no leucine culture.

Jlppropriate samples were taken at 2 min inter

vals for 15 min and the activity of leucyl-tRNA

synthetase was determined. Specific activity is

expressed as/fmoles of leucine attached to

tRNisk/hr/mg protein.

18

SPECIFIC ACTIVITY

,0 CVJ O.

to cvioq

cviis

CM<0

cvi

SPECIFIC ACTIVITY

cvi cvi

cvi

oo

CVI ^z

o S

UJ

CVI

00

cvi

O

Fig. 4

19

(50/fg/ml) to no leucine, the cells were taillipore filtered

from the original medium then transferred to a no leucine

medium. Samples were taken from this culture every 30 min

and the results indicated that the derepression of the rate

of synthesis and inactivation in activity of the leucyl-tRNA

synthetase occurred, but derepression was of sufficient rate

to allow a net increase in measurable activity of the enzyme

(Fig. 5). The rate of derepression occurred at about twice

the rate of inactivation, therefore the net change was that

of derepression.

The results of a study of the activity of leucyl-tRNA

synthetase over a two day period of incubation can be seen

in Fig. 6. The specific activity at the onset of the experi

ment, being 0.196, only decreased to 0.193 after 2 days of

incubation. The data suggest that the rate of synthesis and

inactivation of this enzyme were balanced for the duration of

the incubation period employed in this study.

Percent Charging of Leucyl-tRNA

The strain AB1132 was grown in batch culture containing

150/Cg/ml of leucine and transferred to a no leucine culture.

Fig, 5. Effect of leucine restriction on the specific

activity of leueyl-tRNA synthetase for milli-

pore filtered cells of strain AB1132, Cells

were grown in excess leucine and raillipore

filtered from the original medium then trans

ferred to a no leucine medium* Appropriate

samples were taken at the indicated time and

the activity of leucyl-tRNA synthetase was

determined* Specific activity is expressed

as/^moles of leucine attached to tRNA/hr/mg

protein*

SPECIFIC

ACTIVITY

m

too

Fig, 6« Effect of leucine restriction on the specific


days of incubation. Cells were grown in ex

cess leucine and transferred to a no leucine

culture. Samples were taken at the indicated

time and the activity of leucyl-tRNA synthe

tase was determined. Specific activity is

expressed as/tooles of leucine attached to

tRNA/hr/mg protein.

O

SPECIFICACTIVITY

_.

i_—

ro

ro

K)

22

Samples were taken at 6 and 12 min time periods. The tRNA

was precipitated and divided into a control and sodium

periodated treated sample. Sodium periodate cleaves the

bonds that form cis-hydroxyl groups of ribose of leucine

tRNA, except when leucine is attached to 3* OH groups of the

ribose sugar. As shown in Fig. 7, the percent charging of

leucine tRNA (in. vivo) was essentially the same for both con

trol and treated sample. This result suggests that the change

in rate of synthesis (derepression/repression) and inactiva-

tion of activity of leucyl-tRNA synthetase was not tightly

coupled with the rate of acylation of leucine to leucine

tRNA.

Fig. 7. Comparison of percent charging of leucine to


treated sample. The strain AB1132 was grown

in batch culture containing 150>5rg/ml of leu

cine and transferred to a no leucine culture.

Samples were taken at a 6 and 12 min time

periods. The tRNA was precipitated and

divided into a control (•-*•) and sodium

periodate treated sample

23

o

9000-

8000"

7000-

6000-

5000"

4000-

0 4 6

TIME (MIN)

Fig. 7

8

—r~

10

—T"

12

CHAPTER V

DISCUSSION AND CONCLUSION

The results presented in this study provide evidence

that the synthesis of leucyl-tRNA synthetase is not **con~

stitutive" in nature but is regulated by a leucine mediated

repression.

Nass and Neidhardt (1967) reported that restriction of

either phenylalanine or isoleucine supply to the cells

caused a derepression of their respective rates of synthesis.

From these results, however, they were still unable to con

clude that restriction of appropriate amino acid supply is

sufficient to derepress other synthetases. Furthermore, in

this study, it was shown that although inactivation occurred

during leucine restriction, derepression in the rate of

formation of the leucyl-tRNA synthetase did occur.

As suggested by Nass and Neidhardt (1967), there is

always a possibility of there being multiple synthetases for

a given amino acid. Yu (1966} has reported that there might

be multiple leucyl-tRNA synthetases in E. coli^. This could

be a probable explanation for the observation that dere

pression in the rate of formation of the leucyl-tRNA

synthetase is apparently followed by inactivation of the same

enzyme activity during prolonged leucine restricted growth.

24

CHAPTER VI

SUMMARY

The results of experiments reported in this thesis are

summarized as follows:

1. Synthesis of leucyl-tRNA synthetase is not "consti

tutive1* in nature but regulated by leucine mediated

repression.

2. Readdition of leucine to an auxotroph caused an in

crease in the leucyl-tRNA synthetase activity by

halting the turnover of this activity during

leucine restriction.

3. Leucine restriction caused a two-fold inactivation

of the leucyl-tRNA synthetase activity.

4. The percentage charging of leucine to its specific

tRNA is not specifically correlated with the rate

of formation of leucyl-tRNA synthetase and level of

exogenous supply of leucine to the cell.

25

LITERATURE CITED

Ames, B. N., and B. Gary. 1962. Histidine operon. pp. 322-342.

In B. N. Ames and P. E. Hartman, (ed.) The molecular basis

of neoplasia. University of Texas Press, Austin.

Eoraan, H. G., and I. A. Boman, and W. K. Maas. 1961. Regulation

of OTC and arginine activating enzyme in Bscherichia coli.

pp. 297-304. In I. W. Goodwin and O. Lindberg, (ed.)

Biological structure and function. Vol I. Academic Press,

New York.

Praenkel, D. G., and F. C. Neidhardt. 1961. Use of chloram-

phenical to study control of RNA synthesis in bacteria.

Biochem. Biophys. Acta 53s96.

Lowry, D. H., and Rosebrough, N. V., Farr, A. L., and R. J.

Randall. 1951. Protein measurement with the folin-

phenol reagent. J. Biochem. 193:265.

Nass, G., and F. C. Neidhardt. 1967. Regulation of formation

of aminoacyl-ribonucleic acid synthetase in Sscherichia

coli. Biochem. Biophys. Acta 134s347.

Williams, L. 5., and F. C. Neidhardt. 1969. Synthesis and

inactivation of amino acyl-transfer RNA synthetase

during growth of Sscherichia coli. J. Mol. Biol. 43s529.

Yu, C. T. 1966. Multiple forms of leucyl-tRNA synthetase of

Bscherichia coli. Cold Spring Harbor Symposia on Quanti

tative Biology. Vol. XXXI. pp. 565-570.

26

ABSTRACT

BIOLOGY

WASHINGTON, HAROLD L, B.S., Clark College, 1968

Repression of Leucyl-Transfer Ribonucleic Acid Synthetase of

Sscherichia coli

Advisor: Dr. Luther S. Williams

Master of Science degree conferred June 1, 1970

Thesis dated June, 1970

This study was undertaken to determine whether the for

mation of leucyl-transfer ribonucleic acid synthetase (tRNA

synthetase) was "constitutive11 in nature or was subjected to

some control mechanism. The growth of wild type cultures in

minimal-glucose medium supplemented with excess leucine, as

compared to minimal-glucose medium, did not affect the

specific activity of the leucine synthetase. Using an auxo-

troph for leucine, a shift of this culture from a medium

containing excess leucine to restricting (growth rate)

amounts of leucine, resulted in a significant increase in the

specific activity of the leucyl-tRNA synthetase for the

first hour of incubation. This indicated a derepression in

the rate of formation of the enzyme. After the first hour of

incubation, there was a decline in the rate of synthesis and

some inactivation of this synthetase activity. Further

studies provide results that the addition of leucine to the

highly derepressed cultures caused repression of synthesis

and halted the inactivation of the leucyl-tRNA synthetase.

These results confirm previous findings indicating that the

rate of formation of arginyl- and histidyl-tRNA synthetase

was regulated, and suggest that the regulation of aminoacyl-

tRNA synthetase formation follows a repression-like

mechanism.

SPECIFIC

ACTIVITY

ot\>

Ui

<J\

O)

-4

CD

<O

I1

I—

c

-n

ro-

O

ai.

o

rn

i

m•

m o

11

Table 1. Effect of L-leucine limitation on specific activity

of leucyl-transfer ribonucleic acid synthetase of

strain AB1132.

Sample (hr|

1

2

3

4

5

Condition

Leucine

limited

it

ti

Excess

leucine

u

CPHa

2044

588

424

1536

1598

CPMb

511

147

106

384

397

Protein (rag)

0.0025mg

0.0015

0.0021

0.0038

0.0038

S.A?

0.8

0.39

0.201

0.404

0.421

CPU refers to counts per hour of L Cj leucine attached totRNA.

CPM refers to counts per min of L14cJ leucine attached totRNA.

c

S.A. refers to specific activity as expressed as moles of

leucine attached to tRNA per hour per mg of protein.

12

several generations. Upon transferring the cells to leucine-

free medium, appropriate samples were taken at specific

intervals and assayed for leucyl-tRMA synthetase activity.

As shown in Table 2, synthetase activity increased from

1.13 units/mg protein during growth in restricted culture to

2.74 units/mg protein for the first hr of incubation. However

as samples were taken for the remainder of the 5 hr of the

incubation period, the specific activity decreased to 0,77

units/mg protein. This shift from a leucine culture to a no

leucine culture caused the activity of the leucyl-tRNA syn

thetase to be inactivated. The rate of formation of the

leucyl-tRNA synthetase during derepressive growth conditions

and inactivation of activity is shown in Fig. 2.

The significant observation made in this study of the

auxotroph was the fact that leucine restriction resulted in

an inactivation of formation of leucyl-tRNA synthetase

activity. To ascertain the state of leucyl-tRNA synthetase

during each hour of incubation, it was necessary to perform

similar studies using this leucine auxotroph.

The strain was grown in batch (minimal medium containing

leucine, 150Ag/ml) culture for several generations. Upon

13

Table 2, Effect of leucine deprivation on the specific


synthetase.

Sample (hr)

0

1

2

3

4

5

Condition

Excess

leucine

No

leucine

it

u

it

ii

CPHa

1348

1918

1466

1548

334

538

CPMb

337

479

366

389

83

134

Protein (mg)

0.012

0.0007

0.0009

0.0011

0.001

0.0007

S.A?

1.13

2.74

1.63

1,36

0.334

0.77

CPH refers to counts per hour of

tRNA.

CPM refers to counts per rain of

tRNA.

C14 1, Cj leucine attached to

Cjleucine attached to

"S.A. refers to specific activity as expressed as/fmoles of

leucine attached to tRNA per hour rag of protein.



AB1132. Cells were grown in excess leucine

overnight and shifted to a no leucine

culture. Appropriate samples were taken at

the indicated time and the activity of

leucyl-tRNA synthetase was determined. Spe

cific activity is expressed as .Amoles of
