3 March 5 Number 1978 Volume Volue 5 umbr 3 arch197

Nucleic Acids Research Nuceic

cid

Resarc

The effect of specific structural modification on the biological activity of E.coli arginine tRNA T.A. Kruse and B.F.C. Clark Division of Biostructural Chemistry, Institute of Chemistry, Aarhus University, Aarhus, Denmark anc M. Sprinzl Max-Planck-Institut fUr Experimentelle Medizin, Abteilung Chemie, G8ttingen, GFR

Received 5 January 1978

ABSTRACT Escherichia coli arginine tRNA, has been modified at position s2C32 with iodoacetamide and a spin labelled derivative. The small effects on the charging ability of tRNA by the modifications suggest that the synthetase does not bind to the tRNA in this region of the anticodon loop before the anticodon. A ternary complex of elongation factor Tu, GTP and the modified ArgtRNA, can be formed allowing future studies of enzymatic binding to the ribosome. Using the triplet binding assay the native Arg-tRNA, decodes all 4 codons beginning with CG. The modified Arg-tRNA, has a restricted decoding but the decoding pattern is still unusual according to the Wobble Hypothesis.

INTRODUCTION Now that a three-dimensional structure is known for yeast phenylalanine tRNAI'2 and this is believed to be generally correct for other tRNA species, there is considerable enthusiasm for attempting to relate the chemical structure with the biological activity of this group of macromolecules. Furthermore the information gained from a study of how tRNA molecules interact with and recognize other nucleic acids and proteins will probably have wider applicability in the general field of gene expression where larger RNAs are involved. Within the general context of attempting to relate structure and function of tRNA we have chosen to study the biological effect of chemically modifying specific locations in particular tRNA species. The present work described herein, using monofunctional reagents although complete in itself is preliminary to.work involving bifunctional reagents using the same tRNA site as in this work for crosslinking to complexed proteins. We have also used a derivative of our standard reagent iodoacetamide (Fig. 1B) containing a spin label3,4 for electron spin resonance studies which might give information on whether the tRNA changes shape during interaction with a protein. These reagents have Abbreviations used: acm = carbamoylmethylSL = spin label C Information Retrieval Limited 1 Falconbeg Court London WI V 5FG England

879

Nucleic Acids Research also been used in a companion study I on yeast tRNAPhe in which s2C has been enzymically introduced. Our general approach is to test the modified tRNA for several biological functions. The E. coli tRNA used in these experiments is tRNAl 9 5,6,7 (Fig. 1A) because it contains one 2-thiocytidine (s2C32) residue two bases before the anticodon so this provides our specific modifiable site. The biological activities tested are charging with an amino acid by E. coli arginyl-tRNA synthetase, formation of a ternary complex of modified Arg-tRNA with elongation factor EF-Tu, and GTP, and binding to ribosomes induced by oligonucleotides of defined sequence. MATERIALS AND METHODS Purification of tRN Arg

Unfractionated tRNA from E. coli K12 strain CA 265 was obtained from Microbiological Research Establishment, Porton Down. A four-step procedure was used to purify tRNAArg: 1) BD-cellulose column-chromatogra;hy 8 at 40C using a linear NaCl-gradient from 0.45 M to 0.75 M in a buffer containing 10 mM Tris HCl pH 7.6, 10 MM MgCl2, 2 mM NaN3 and 1 nM Na2S203; tRNAArg was eluted at approx. 0.6 M NaCl, 2) column-chromatography on Sepharose 4B I applying a decreasing (NH4)2SO4- gradient from 1.3 M to 0.7 M in a buffer containing 20 mM NaOAc pH 4.5, 10 mM MgCl2, and 1 flM Na2S203; tRNAArg was eluted at approx. 1.0 M (NH4)2SO4, 3) reverse phase chromatography (on RPC-5) 10 applying a linear NaCl gradient from 0.5-0.6 M in a buffer containing 10 mM Tris HC1 pH 7.5, 10 mM MgCl2 and 2 nmM Na2S203; tRNAArg was eluted at approx. 0.54 M NaCl, 4) rechromatography on RPC-5. Fractions were assayed for arginine acceptor activity according to Holladay et al.11. The major tRNAArg was obtained at a purity of 1500-1600 pmoles/A260-unit, and it reacted with approx. 1500 pmoles 14C-iodoacetamide per one A260-unit tRNA, indicating one mole of s2C per mole tRNA. The sequence of E. coli tRNA is shown in Fig. A and is consistent with the major tRNAArg purified by us although no full sequence analysis was carried out. Cel 1 extracts Ribosomes were prepared 12 from E. coli MRE 600 cell paste obtained from Microbiological Research Establishment, Porton Down. The S 100 from ribosome

preparation was either used as crude E. coli enzyme mixture after 1% streptomycin cut, 70% ammonium sulphate precipitation and batchwise removal of nucleic acids on Sephadex - A 50, or it was used for the preparation of puri880

Nucleic Acids Research A

c C A pG * C C * G A * U U * A C * G C * G G * C c c u CC

s4U C G A D G G

A D A

NH2

*

*

A cu c G ..A.

*

*

GGAGG C

U A A

0

TU)p

0

OH

x cr m7G %a

AAU

A

C U C G G

U

U2c

* * * * *

G

G A

G C C

RI = -CH2-CO-NH2 R2 = -CH2-CO -NH

¶N-O

A I

m2A

A

Fig. 1A: Nucleotide sequences of E. coli tRNAArg 5956_7 * The modification sTte s2C32 -is marked by an arrow.

Fig. 1B: Structure of the s2C-residue after alkylation with iodoacetamide or spin label.

fied ariginyl-tRNA synthetase according to F. von der Haar 13 * The purified synthetase was used for kinetic measurements, the crude enzyme mixture for other assays. Alkyation of

tRNAI-9

14C-iodoacetamide with a specific activity of 57 mCi/mmol was obtained from the Radiochemical Center, Amersham. The spin label 4-(2-iodoacetamido)2,2,6, 6-tetramethyl piperidino-oxyl (see Fig. 1B) was obtained from SYVA (Palo Alto, Ca., USA). Alkylations were carried out at 370C in 10 mM KPO4 pH 7.2 with 1 mM reagent except for the preparative alkylations with the spin label where the reagent concentration was 2 mM. The tRNA concentration was 0.5 mg/ml in analytical experiments and up to 4 mg/ml (, 0.12 mM) in preparative incubations. For preparative purposes the incubation time was approx. 20 hr. The modification was removed by incubating the alkylated tRNA in 50 mM HEPES p1l 7.0, 10 mM MgCl2, 50 mM KC1, 50 mM NH4Cl and 28 mM S-mercaptoethanol or 1,2-ethanedithiol as indicated at 37°C for 4 hr. The tRNA was desalted by 881

Nucleic Acids Research gel filtration and precipitated by addition of 2 vol. 96% ethanol before use. Anal,ysis of -alkyl ation

Carbamoylmethylation of tRNA,rg was followed by use of '4C-labelled reagent. The extent of alkylation was determined as the amount of TCA-precipitable radioactivity, that was collected on 3 MM-filter paper discs and counted by liquid scintillation. The specificity of the reaction was analysed by three different methods: 1) Nucleoside analysis by cation exchange column chromatography as described earlier 14, 2) the extent of alkylation of the s4U-residue in tRNAArg was determined by following the ratio between UV-absorption at 335 nm (characteristic for s4U) and absorption at 260 nm during reaction with iodoacetamide, and 3) native and alkylated tRNA, was digested with RNase T1 and analysed by chromatography on an RPC-5-column (see Fig. 2). 2.25 A260 or 3 A2 6p tRNA (4C-acm s2C32) were incubated at 37°C for 3 hr. with 250 units RNase T1 in 100 pl 100 mM Tris HC1 pH 7.6. The digests were applied to an RPC-5-column (0.5 x 80 cm), and eluted with first a gradient of 250 ml total volume ammonium carbonate pH 9.2 from 0.05 M - 2.00 M, and in order to elute the larger fragments a second gradient 0.1 M - 0.6 M NaCl in 50 mM NH4CO3, 100 ml total volume. Chromatography was performed at room temperature at 16-18 atm pressure, giving a flowrate of approx. 90 ml/hr. Fractions of 2.8 ml were collected and the UV-absorption of the eluant was monitored on an ISCO-UA5 monitor with a Type 6-optical unit attached. In the case of tRNAArg (14C-acm s2C32) 0.5 ml samples were taken from each fraction and counted in Aquasol.

tRNAArg

Assay for determining kinetic_parameters

of the

aminoacylation

of

I

Arg

Different amounts of tRNAArg were incubated at 370C in a reaction mixture containing: 100 mM HEPES pH 6.7, 10 mM MgCl2, 8 mM KC1, 4 mM ATP, 0.24 mM CTP,6.4 uM 3H-arginine HC1, specific activity 11000 mCi/mr,;ol and 1.46 x 10-4 A280 purified arginine-tRNA-synthetase in a volume of 100 p1. The reaction was started by the addition of tRNAArg 20 p1 aliquots were taken after 1', 2', 3' and 4', spotted on 2.5 cm Whatman 3 MM filter discs, that were washed 2 x 10' in 5% TCA and 1 x 2' in EtOH, dried and counted in toluene-scintillation liquid. The extent of aminoacylation was calculated using a specific activity of 3080 cpm/pmole (12.7% counting efficiency) and the results plotted as a function of time. The best straight line was taken as the initial rate of aminoacylation, VO, at the specific tRNA concentration. In all cases less than 25% of the tRNAArg was aminoacylated within 4'. KM- and Vmax-values were 882

Nucleic Acids Research determined from Eadie-Hofstee-plots, i.e. V0 plottet against

Formation of

Vo/[tRNA].

ternary com2l2ex. EF-Tu.GTP-arginyl:tRNAIrg

EF-Tu*GDP was a gift from Dr. D.L. Miller, Roche Institute of Molecular Biology, New Jersey, USA. EF-Tu.GTP was prepared from EF-Tu.GDP in the following way 15: 600 pmoles EF-Tu.GDP (1.5 x 10 5M) were incubated for 15' at 270C with 5 mM phosphoenolpyruvate, 5 x 10 5M GTP, 0.25 mg/ml pyruvate kinase, 50 mM HEPES pH 7.0, 10 mM MgCl2, 50 mM NH4Cl and 50 mM KCI in 40 pl total volume. Then approx. 70 pmoles of the tRNA to be tested were added, the incubation was continued for 5 more min. and the mixture was injected cnto an Ultrogel AcA44-column (0.6 x 50 cm) and eluted at 40C with elution buffer: 10 mM Tris HCl pH 7.5, 10 mM MgCl2 and 100 mM NH4Cl, at X' 60 cm hydrostatic pressure giving a flowrate of 7 ml/hr. 8-drop-fractions were colklcted and 4 ml of BBS-3-scintillation liquidwereadded to each fraction before counting. The sample for measuring the ESR-spectra of EF-Tu bound tRNA contained in 22 }l of the same buffer 0.4 mg ("s 9 nmoles) EF-Tu.GDP, 10 pg pyruvate kinase, 50 nmoles GTP, 400 nmoles PEP and 2.3 A260 (ni 3.5 nmoles) 14C-arginyl-tRNAAr9 labelled with the spin label; all components except the tRNA were incubated at 37°C for 15' before addition of tRNA. In control/txperiments GTP and PEP were omitted in order to avoid phosphorylation of the GDP bound to EF-Tu. After the ESR-measurement the mixture was injected onto an AcA44-column and chromatographed as described above. From each fraction 50 pl were counted in BBS-3-scintillation liquid, the rest was dried in desiccator at room temperature, taken into 25 pl H20 and used for determination of the relative amount of spin label from the height of the central peak in the ESR-spectra. ESR-spectra of charged or uncharged tRNAArg (SL s2C32) were recorded at the same tRNA-concentration and the same buffer as above. Ribosomal

binding

assay

Non-enzymatic triplet dependent binding of native and alkylated arginyltRNA,9 was tested according to Leder 16. 150 pmoles 3H-Arg-tRNAAr9 were incubated with 2.4 A260 ('\, 60 pmoles) ribosomes and 0.15 A260 (". 5000 pmoles) triplet in 100 mM Tris HCl pH 7.2, 50 mM NH4Cl and 15 mM MgCl2 in 50 pl, 2 ml of the ice cold buffer were added, and the mixture was filtered through a nitrocellulose Millipore filter that was washed three times with 2 ml buffer. The filter was counted in Brays solution. Controls were made without the triplets. Different Mg2+ concentrationsand different incubation conditions were tested. 15 nM MgCl2 and incubation for 20 min. at 230C were found to give the best results. 883

Nucleic Acids Research Trinucleoside diphosphates C-G-C, C-G-G and C-G-U were prepared according to Leder 17 C-G-A-A was a generous gift from Dr. J.H. van Boom, Leiden. ESR-spectra were recorded on a Varian E3-spectrometer using a microwave powerof5 mWor less to avoid saturation. The samples having a tRNA concentration of approx. 1.5 x 10 4 M were introduced in glass capillaries of about 1 mm inner diameter. The rotational correlation time, T, of the spin label can be calculated from an expression involving either the linear or the quadratic term of the nitrogen quantum number m 18,19. The following tensor elements are used Az=87 MHZ, Ax=Ay=14 MHz, gx= 2.0089, gz=2.0027 '-, which gives the following expression for T (involving the quadratic term in m): T = 6.5 x 10-1 X ( /ii +

; 2) x AS(o) sec

where AB(o) is the peak-to-peak separation of the central hyperfine component of the derivative spectrum and h(m) is the heights of the peaks. All spectra were recorded four times at 24-260C with scan times of 8-16 min., and the mean values from the four runs were used in the calculations. In no case could any systematic change in the spectra be detected during the four runs. RESULTS As sihown earlier 2-thiocytidine can be alkylated by alkylhalides, e.g. iodomethane or iodoacetamide 21, in neutral or slightly acidic aqueous solutions at low temperatures, (Fig. 1B). E. coli tRNAlAr , was alkylated with iodoacetamide in a pseudo - 1st order reaction having a second order rate constant k=2.2 min-1 M 1. For comparison yeast tRNAPhe, in which the penultimate nucleoside at the 3'-end normally a cytidine is enzymatically replaced by 2-thiocytidine 22 is modified under the same conditions with a rate constant k=3.1 min-1 M 1. Nucleoside analysis showed the disappearance of s2C and appearance of a new nucleoside with the same chromtographic behaviour as acm s2C after preparative alkylation. It was also shown that in agreement with the literature the modification can be removed by certain nucleophiles, e.g. ethanedithiol, giving rise to s2C again, or a-mercaptoethanol, giving rise to C. The specificity of the reaction was analysed by RNase Tl-digestion and column Chromatography on RPC-5, as shown in Fig. 2. Except for a small amount of unreacted '4C-iodoacetamide eluted at the break-through volume, the only 884

Nucleic Acids Research

A

0

°

05 -~ ~ ~ ~ OL03

10

20

30

~

~

50

~

60

70

80

90

100

110

120

130 f ract ion

Fig. 2: Analysis of RNase Ti-digests of A) tRNAArg modified by 14C-iodoacetamide, and B) native tRNAArg by RPC-5 column chromatography. Experimental conditions are described under Materials and Methods. radioactive peak coincides with a new peak in the U.V. absorption-profile. From the nucleoside analysis of this peak shown in Fig. 3, it is evident that it contains I, U and acm s2C in nearly equimolar amounts as expected for acm s2C-U-Ip T1-fragment. In addition it contains G and smaller amounts of A and C, the sum of the latter equalling the amount of G, as expected when some of the dinucleotide peak (containing ApGp and CpGp) is pooled together with the alkylated trinucleotide. From following the decrease in U.V.-absorption at 338 nm during alkylation and from determination of the amount of radioactivity bound to tRNA9 that is not removable by 0-mercaptoethanol treatment, it can be concluded that the extent of alkylation of S4U under standard conditions is about 4%.

Aminoacylation

of modified tRNAAr_ Carbamoylmethylation of s2C or conversion effect on the extent of aminoacylation (Table has no effect on the kinetics of the reaction s2C causes a 2-fold decrease in KM and a 2 to

of s2C to C in Ar has no 1). The s2C to C-conversion either, whereas alkylation of 3 fold decrease in Vmax' 885

Nucleic Acids Research

.0a.

QOlL

0~~~~~~~~

C4

~~5

i

10

25

50 75 Elution volume (ml)

Fig. 3: Nucleoside analysis of the radioactive Ti-fragment (Fig. 2A) 14. Aminoacylation pmoles/A260-unit

KM nM

Vma, rel

1680

75

100

1720

37

52

tRNA,9 (SL s2C32)

1650

35

33

tRNAArg tRNAArg

+

1630

70

100

(acm s2C32)+

1640

66

94

tRNAIArg

(SL S2C32)+

1605

73

95

tRNA species

tRNA,r9

|tRNAArg

(acm s2C32)

Table 1: Kinetic parameters for the enzymatic aminoacylation of native and modified tRNA rg. +tRNA treated with S-mercaptoethanol as described under Materials and N2thods. Formation of the

ternary

complex

EF-Tu-GTP.arginXl:tRN Arg

Fig. 4A, B show how gel filtration on an AcA44-column can be used to separate free tRNA and tRNA bound to EF-Tu-GTP. Carbomoylmethylation and spin labelling of Arg-tRNAArg does not prevent formation of the ternary complex with EF-Tu.GTP (Fig. 4C, D). It is evident from Fig. 4 that neither the "4Calkyl group nor the spin label is released under the conditions of analysis, as that would give rise to 14C-counts and spin label eluting together with the small amount of free arginine eluting about fraction 32. It is also evident that a small proportion (