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The undersigned certify that they have read, and recanmend to the Faculty of Graduate Studies and Research, for

140 phenylalanine Incorporation

acceptance, a thesis entitled, by Wheat-Seedling Cytoplasmic Ribosomes", submitted by Edward B. L. Tucker, in partial fulfilment of the requirements

for the degree of Master of Science.


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V4 e_phenylalanine incorporation was induced by homogeneous

preparations of wheat-seedling cytoplasmic and chloroplast ribosomes which had been isolated by zonal centrifugation. Wheat embryo

synthetase enzymes in the presence of stripped yeast tRNA and a

C-phenyl- alanine produced Mae -phenylalany] tRNA which was used in the incorpora- tion systen. The incorporation by cytoplasmic 79S ribosomes required high concentrations of ribosomes, high concentrations of magnesium, low concentrations of tris-HCl buffer pH 7.6, and was complete after 45

minutes incubation at ave This transfer of 4


C-phenylalanine fron C-phenylalanyl tRNA into protein was not dependent upon added energy factors. Incorporation by both species of ribosomes was inhibited by puromycin, only the chloroplast ribosomes were inhibited by chlor- anphenicol, and neither species of wheat-seedling ribosomes was inhibited by cycloheximide. The cytoplasmic ribosomes dissociated into subunits which would reassociate to form the parent species. Puromycin pretreatment of ribosomes resulted in complete dissociation. The reformed parent species would not induce polyphenyialanine synthesis. The results led to the conclusion that the honogenization and separation precedures had cleaved the polysomes into monosomes which contained

not only mRNA fragments, but nascent protein as well.

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The research described in this thesis was performed under the supervision of Dr. Saul Zalik. His guidance, wisdom and assistance was given willingly throughout the study and in the preparation of this manuscript.

I wish to acknowledge my colleagues for helpful criticism and direction during the research; and Dr. Sara Zalik (Department of Zoology) for the use of equipment.

Appreciation is given to my wife, Lindis, for her encouragement and helpful discussion,

The financial assistance was provided by the University

of Alberta and the National Research Council of Canada.

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II. Differences between Chloroplast and Cytoplasmic Prove eo Vines | Zt UM GOUD LCAae taigttee cenit wetn enc + << 6s 9 Li eeaiinoeAGldsincoroonaciOn DY Plant SYSLEMS, 9% 7... «ess se 1 A. Amino acid incorporation by wheat 80S ribosomes ., 12 B. Amino acid incorporation in other higher plants .. 17

C. Amino acid incorporation by chloroplasts and lu Olao live scam yal Ds CINE S mative rate amace ie rer 8 cle tet ses aia! 6 20 1. Amino acid incorporation by chloroplasts ....... 20

2. Amino acid incorporation by chloroplast ADU S ANG Semmerere taste as atete sche cia grate are coher eht el ete at nis i 22 IVa BLISSUCTULlUNsULebUKaryOte RI DOSUNGSE tps... cc cnc nie siecs es A. Dissociation of ribosanes by high salt

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Checking Chemicals used in the Incorporation Reaction. ee LACTIS, <P isn eee oer eee

B. Creatine phosphokinase

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Chenical Preparation

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Bend LOSCULITGL IG mmraee prere ie ate gen Or er eR a CUR OLS Ciel aLONmO MeDiEllO Way Gir ai eile te we dcten teria D. Preparation of chromatographic matertal ........... ECC LICR EU eet tins Si ais ¢ Me UACE ERD Gees S eatece se CES COU OMSK EG OD Cir cic dee suis. ginid tats sheik ofa, sats saale 010 wes vis

E. Treatment of sucrose with diethyl pyrocarbonate prem Li © | OMMreretet sista dtetrcerg cesta ste 5 ara,s% ele ee eval e catheters ere 4 Preparation of and Procedures with Ribosomes ........ PE CCU LeM id \MMPM ae a ritate esis crs ey sen ke Cartes cine aes SL SU La CIO MEOIMRTOUSONES Mare grils ccinip piers cc aisle sete aiace lacs TERT DOSONeSEOr ZONaLesehardc1 ON sata ce sci csece’s «eas Cap RiDOSOMmeS WILMOUL-ZONd! S@ardalion ...2ecrsess«

C. Separation of ribosomes by zonal centrifugation ...

D. Dissociation of ribosomes and separation of

SU DUNE Summer un tir aercitee ettre vis iysierae Reeetete ahersie seetaeainas ene ie.s Bemeova nc ano sDUCKALECeENL Yt TUdd CHOM a. leteeicias suis «i9ia'e-s POPLULONVCLUMLEE Aue DOL aGIOUSUIICS (.<icre sawaa ceess «ss

1. Preparation of ribosomes for dissociation AOWN TOOL UUCKGLS) Gates sane aes cele acaeuipe a) ses ers

2. Preparation of ribosomes for zonal dissociation.

G. Estimation of RNase in prepared ribosomes ........ He @OCOIEN Lot OMOVAIUGS. “vccguesnt ys s Mey wees areata de SR@SC UG sce De eee ie di atime wets Co et ee

2. Calculating sedimentation EU ie ree Serene

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iio BP OLedUreceeLOVMCOePOY AL] Ol. vss o0s soe ss age selene wese A. Preparatton of synthetase enzymes ........cscecsees B, Preparation of ribosomes from wheat germ .,........ C. Preparation, of 4c phenylalany] CRNADBeGe et .* SR eas LPMELELCOCDOYe Witimncac L 1OMMMy 4 wit es gates ote gic actress ae ome CEDORG GhOLMU MME OGLOE 0s; gulp 'g sare pain sas cree steeren ee RESULTS AND: DISGUSSIUNSSray. and.e ceva santos . fort ener esc 03 : ee SOLACLONS OTR DOSONGSaar, as, Se TOON EE Sameera A. Characteristics of zonal separated ribosomes .,... B. Ribonucleases in zonal separated ribosomes .,,..... II. Phenylalanine Incorporation by Zonal Separated RIDOSONeSeUs INOmdal Gans Terao YS LOMem, yigutcmie far tea er Biwi DOS OMNES CONCEMCAL1 ONT ©. irre ely ste sce acaltee ine ales es Be weLNCUDALIONECIMNG TOF aNCOFDOratl ON wncinaiaes se < sees « (ee bUrreneer coc benim NCOVDOY ALTON er fy a ose ware sss write D. Dependency of incorporation upon added energy ROCUOVS oem ire ciel gheta seus nee © ie seats Peer Cor Eber Cem diecOl canind bLON ame c iis ce oe Pee Oe F. Rate of incorporation as a function of magnesium COME CMU tral) Ol mmepeee eter eens a sie ete a tee iiiaees fevers slats ty ecstatic G. Effect of inhibitors of protein synthesis on TIGL Om aia Ol emmmme neattteisrettc sr sincere sontetaeleig 00 ie Se verdaa es ely H. Species specificity of tRNA and synthetase enzyme . III. Dissociation Characteristics and Incornoravion with

Zonal Separated Ribosomes ,......., Ae tr Pca RCC

Dime 1 aS Game etter cs sie hive a ane pie os aE ee re Mee UTSSUCTALRON MMe crip e tee ts cua ns ak vere w me Kors ue 3 2. Incorporation with recombined monomers een B, Puromycin otP. bw. £86. 8 eS ee PTA ted Ne) Re MS Ae O14 £2 Bee SGC e RS Pe 88 2) Fe eee DIS SOCTACA ON Maras sateen Care chat sas om etd

2. Incorporation with recombined monomers ......





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Effect of buffer on incorporation of 140 oheny1- alanine by cytoplasmic wheat leaf ribosones

Requirement of energy factors for incorporation by cytoplasmic wheat leaf ribosomes ............

Effects of chloramphenicol, cycloheximide and puronycin on incorporation by 70S and 80S FIDOSOT es mi COleWied tas CAVES Ae. he as ae sos ce cele

Incorporation by subunits of high salt dissociated SUS svied emled GelhbOSONCSEme rT. 2.0.11 cite cis ict ce.

Incorporation by subunits of high salt dissociated 80S wheat leaf ribosomes which had been pretread- SW Lila OLICONY Cal TMM ce Meas action tots cats ue fols sb cadiede io <oeeesete

Factors required for incorporation by washed WEA Lealic dla DOSONC Sie an hiusueceaie eeiii als noe wieatiew:«

Effect of addition of partially purified super- natant factor on the incorporation by NH C1 WaShedsWhedlealPateriDOSOMNGS Boas ass ccs esse see sae

Incorporation by wheat leaf ribosomes fran seedlings of varying ages .,.,. A ae ENS Sgr RRA lee

Incorporation by subunits of high salt dissociated (300 m KC1), 80S ribosomes obtained from 3.5 day-old wheat seedlings and pretreated with

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The mechanism of protein synthesis ..,,..... feiss

The structural relationship of ea to an amino acylated tRNA ......... Penn a ak eer

Analytical ultracentrifuge pattern of leaf ribosomes. from Manitou wheat ",..9,....02..6.<..-

Zonal separation of wheat leaf ribosomes .......

Analytical ultracentrifugation pattern of zonal separated 70S and 80S ribosomes fron wheat leaves

Assay for RNase on wheat leaf ribosomes ........

Zonal separation of wheat leaf ribosones through ceUEE Urea ceUsuu tL hC ramet rr Nis oor tees mts. went aye

Activity versus concentration of 80S wheat leaf EIDOSONG SUM Bree scl r dt a ta al dk eee date te ede

Wosphenylalanine incorporation by cytoplasmic wheat leaf ribosomes versus time .,......ee0- he

Effect of tris concentration on the phenylalanine incorporation by cytoplasmic wheat leaf ribosomes

Effect of magnesium concentration on M40 pheny1- alanine incorporation by the 80S wheat leaf PIBCSONESIC HON, VRINNS. OF i ant, MEPS, FI DSS eS...

Comparison of the chloroplast ribosomal rate of incorporation as a function of magnesium concentration with that of the surrounding

CY COP LasnriGri DOs ONES! OT MWNEAT S «cert sect tat alate! ake

Analytical ultracentrifugation pattern of 80S wheat leaf ribosones suspended in Buffer IV

High salt dissociation of 80S wheat leaf ribosane

Puronycin stimulated dissociation of wheat leaf CYCLOP TAStC Mi DOSONGS= =... ar a Pigs relarieats eiaca ate aie








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Figure Page

16 Effect of various concentrations of KC] on the

dissociation of puromycin treated 80S wheat

LES tem sChGGmene Weer ti sk, te eee Pies rene 84 17 Zonal dissociation and separation of 80S wheat

leaf ribosomes pretreated with puromycin ....... 86 18 Reassociation of subunits of wheat leaf cytoplasm

cytoplasmic ribosomes ...,..... SBR nae iene 88 19 Sedimentation values of ribosanes prepared by

homogenizing wheat leaves in Buffer III. ........ a7 30 20 40 phenylalanine incorporation by washed wheat

leche DOsOlesmVercUS aU IMGscser ai ai, eet lt ba 93 21 Analytical ultracentrifugation conparison of

ribosones prepared from wheat leaves of different

DUES eS MAM ee tore ue itean ae ee Ce eee er Pena, 96 oe A comparison of the ability of ribosomes, fron

different ages of wheat seedlings, to dissociate

Teil dace lee see a eS Re Tee eee 98 23 Puronycin stimulated dissociation of ribosomes

prepared fron 3.5 day-old wheat seedlings ...... 101 24 The dissociation effect of various concentrations

of KCl on the ribosones fron 3.5 day-old wheat seedlings, treated or untreated with

DUT meme te ee Sar ial ete a ner ae iter 25 Sedimentation values of NH,Cl washed ribosomes

and their subunits from 3, g day-old wheat

SENG See ETI CACC Es eae 105 26 Zonal dissociation and separation of ribosones

froneashedaysoldyseadiings chlerenlest.and cee’ 107 27 Reassociation of and cation effect on ribosome

subunits from 3.5 day-old wheat seedlings ...... 110

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Protein synthesis in eukaryotes is thought to occur by a system generally similar to that of prokaryotes. Dissimilarities in their ribosones and in the initiation of protein synthesis by then have been reported. Leaves of higher plants contain abundant amounts of the two classes of ribosomes in the same cell. ‘Therefore, despite the difficulties associated with sorting out homogeneous ribosome : classes, they afford a unique opportunity for comparative studies.

The ribosomes of the plant cytoplasm are of the eukaryotic class, while the ribosomes found in the chloroplast and mitochondria have characteristics similar to those of prokaryotes. These three types of ribosones are apparently capable of synthesizing proteins. Lately,

a great deal of research evidence has confirmed that the ribosomes found in the cytoplasm are different from those found in the chloroplasts

or mitochondria. Various methods used to obtain relatively-hono- geneous preparations of ribosomes have included organelle isolation, plant etiolation and the use of seed germ. A different approach in studying protein synthesis of the individual types of ribosanes involves the use of specific inhibitors of protein synthesis. In the present study the approach was to separate the chloroplast and cytoplasmic ribosomes fron an heterogeneous mixture by means of zonal centrifugation. Using this method relatively-large quantities of chloroplast and cytoplasmic wheat leaf ribosomes were obtained and the intactness of these was demonstrated by their ability to incorporate 14 0_phenylalanine.

This study was undertaken to characterize the general

features of an incorporation systen containing these ribosomes.

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Experiments on the factors required for incorporation, the effect of protein synthesis inhibitors, and ribosomal dissociation and reassocia-

tion were performed.


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I. Mechanism of Protein Synthesis

A. Introduction

Although the procedure involved in the organization and formation of a protein is a dynamic process, it is often divided into three steps--initiation, elongation, and termination--for ease in comprehension. This section of the literature review is similarly divided; however, Figure 1 attenpts to show protein synthesis in a more operational state.

Boulter (1970) states that "most of our knowledge of the mechanism of protein synthesis is derived fron experiments using 70S

microbial ribosomes, particularly those of E. coli". This portion of

the literature review, therefore describes the process with data obtained fron bacteria rather than from plants. As is discussed tater in this review, protein synthesis in eukaryotes is thought to occur by a mech- anism similar to that for prokaryotes. However, a statement made by Mahler and Cordes (1966) seans relevant here. "The very fact that a relatively simple, unified, and self-consistent model appears to have energed from these efforts might suggest that perhaps some of the gen- eralizations are overly facile and that once the problems are rexanined in search for quantitative rather than qualitative agreement, inconsis- tencies may emerge that may gnaw at the very foundation of the

magnificient edifice”.

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B. Initiation

The mechanism of protein synthesis has been comprehensively described in reviews by Boulter (1970), Kaji (1970), Lucas-Lenard and Lipman (1971) and Boulter, Ellis and Yarwood (1972). It is generally believed that methionyl tRNA is the "universal initiator" in that both eukaryotic and prokaryotic cells contain two species of met tRNA, one of which is used exclusively for initiation of protein synthesis while the other is used exclusively in the elongation step of protein synthesis. However, in the prokaryotic cells, formylated met tRNA is required for initiation while in the eukaryotic cells, the initiator met tRNA is not formylated. As Boulter et al. (1972) have summarized: " (a) micro-organisms contain a transformylase and a fommylatable initiating tRN Uh, (b) animals and yeast do not contain a trans-