Protein-nucleic acid interactions in Xenopus laevis Oocyte 5S Ribosomal RNA gene transcription

(1)

Protein-Nucleic Acid Interactions in Xenopiis laevis Oocyte 5S Ribosomal RNA Gene Transcription by Wei-Qing Zang B. S., Northwestern University, 1982 M. S., Northwestern University, 1987

A Dissertation Submitted in Partial Fulfillment of the Requirements for the Degree of

DOCTOR OF PHILOSOPHY

in the Department of Biochemistry and Microbiology

We accept this dissertation as conforming to the required standard

Dr. Paul^^Rdrudhiulc. Supervisor (Department of Biochemistry & Microbiology)

K Q j^Q ^^^ffc»eD artm ental Member (Department of Biochemistry & Microbiology)

Dr. E dw ard E. Isrfiguro, D epartm ental M ember (Departm ent o f Biochem istry & MicrobioloevY

Dr. Trevor J. Trust, Departmental Member (Department of Biochemistry & Microbiology)

Dr. Nancy St&rwood, Outside Member (Department of Biology)

Dr. ^6el M. Gottes'feld, External Examiner (Dept, of M olecular Biology, The Scripps Research Institute)

(2)

Supervisor: Dr. Paul J. Romaniuk

A B S T R A C T

The focus o f this Ph.D. project was on the in vitro studies o f three protein-nucleic acid interactions itl the X en o p u s oocyte: TFIiTA-5S rRNA gene, p43-5S rRNA and ribosomal protein L5-5S rRNA. The binding properties and contact sites of the proteins on the nucleic acids were determined.

For studying the interaction between TFIIIA and the 5S rRNA gene, a series o f substitution mutants o f Xenopus laevis TFIIIA were constructed and expressed in E. coli strain BL21(DE3). The apparent association constant for each TFIIIA mutant was measured using a nitrocellulose filter binding assay. Th„ results indicate that substitution o f fingers two, three, and four to six o f TFIIIA significantly reduce interaction with the 5S rRNA gene. The fact that substitution of finger three alone reduced DNA binding activity about 100 fold suggests the importance o f this finger to the free energy o f DNA binding. In contrast, substitution o f fingers one and seven had little effect on TFIIIA interaction with the 5S rRNA gene. Furthermore, the results of scanning substitution mutagenesis within the third finger of TFIIIA suggest that contacts made by the a-helical region of finger three provide the majority o f the free energy of the protein-DNA interaction. The results o f single amino acid substitution mutagenesis within finger three reveal, that many o f the DNA binding amino acids are clustered along one face of the a-helix of finger three, which might account for the importance o f this finger to the free energy o f DNA binding. The results presented here indicate that zinc fingers two to six are required for high affinity DNA binding, and the contacts made by the a-helical region of finger three provides the majority of the free energy of the protein-DNA interaction.

For investigation of the RNA binding properties of Xenopus leavis p43, p43 cDNA was cloned into an expression vector, pET-1 lb, and expressed in E. coli. The purified p43 was characterized using a nitrocellulose filter binding assay. The experimental conditions necessary for in vitro p43-5S rRNA complex formation include: pH 7.5, 0.1M KC1 and

(3)

incubation at 22 °C. Under these conditions, the protein binds to Xenopus oocyte 5S rRN A with an apparent association constant of 1.61+0.12 X 10^ M 'V A series of mutations in 5S rRNA were used to determine which sequence and structural features o f the 5S rRNA are required for high affinity binding of p43. The primary' contact points for p43 include the sequences and structures of stems II, IV, V arid loop D of the 5S rRNA. Although p43 and TFIIIA are structurally similar and are both relatively insensitive to mutations in the 5S rRNA, they do require different features o f the 5S rRNA molecule for high affinity binding.

The affinity of recombinant Xenopus ribosomal protein L5 for a set of 5S rRNA mutants was quantitatively measured using a nitrocellulose filter binding assay. Unlike TFIIIA, L5 was insensitive to changes in either the sequence cr the secondary structure of the 5S rRNA. The binding sites of L5 on wild type and three mutant 5S rRNAs were studied using a RNase footprinting assay. The results from the RNase footprinting assay indicated that the binding site o f Xenopus laevis ribosomal protein L5 is dispersed widely along the entire 5S rRNA molecule. The interaction of L5 with 5S rRNA may be dependent on the overall contribution of multiple contacts, rather than any particular site.

The experimental data from these studies indicate that the specific protein-nucleic acid interactions in the storage and transportation o f 5S rRNA, as well as 5S rRNA gene expression, use distinct mechanisms.

Examiners:

if. .

Dr. Paul J. Romaniuk, Supervisor (.Department of Biochemistry & Microbiology)

V

(4)

---5---Dr. Edw ard E. Ishiguro, D epartm ental M em ber (D epartm ent o f B iochem istry & Microbiology)

7 ^ ---Dr. Trevor J. Trust, Departmental Member (Department of Biochemistry & Microbiology)

Dr. Nancy Sherwood, Outside Member (Department of Biology)

" £/ I

Dr. Joel M. Gottesfeld, External Examiner (Dept, o f M olecular Biology, The Scripps Research Institute)

(5)

T able 2 . 1 The effects of TFIIIA finger substitution mutations on binding activity to the 5S rR N A gene and 5S rR N A ...65 T able 2. 2 The effects of TFIIIA scanning substitution mutations on binding activity to the 5S rR N A gene and 5S rR N A ... 66 T able 2.3 The effects of TFIIIA single amino acid substitution mutations within finger three on binding activity to the 5S rRNA gene and 5S rRN A ... 69 T able 3.1 A comparison of the specificity of RNA binding by p43 and TFIIIA 91 T ab le 3.2 Comparison of the relative binding affinities ol TFIIIA and p43 for 5S rRNA m u ta n ts ... 101 T able 4.1 Relative L5 and TFIIIA Binding Affinities for 5S rRNA M utants 122

(10)

X L IS T O F FIG U R ES

F ig u re 1.1 C is-acting elem ents in TFIIIA prom oter...4

F igure 1.2 Amino acid sequence o f transcription factor IIIA from X. laevis oocyte 6 F ig u re 1.3 Two-dim ensional folding scheme for a linear arrangem ent o f repeated do m ains o f T F IIIA ...7

F igu re 1.4 Structure o f the Xfin-3>\ zinc finger derived from NMR studies... 9

F igure 1.5 Organization of the repeating unit in X. laevis 5S D N A ... 12

F igure 1.6 The structure o f Xenopus laevis 5S rRNA genes...14

F igure 1.7 Differential transcription factor association with oocyte and somatic 5S rRNA genes influences transcription during developm ent...17

F igu re 1.8 Model for gene programm ing during chromatin assembly on a 5S rRNA g e n e ... 18

F igure 1.9 Model for differential oocyte and somatic 5S rRNA gene expression... 21

F igure 1.10 Schem atic representation o f 5S rRNA secondary structure... 24

F ig u re 1.11 Secondary structure o f the 5S rRNA from E. coli ... 25

F igure 1.12 Proposed Xlo m ajor oocyte 5S rRNA secondary strrctural model...27

F igure 1.13 The secondary structure o f 5S rRNA from Xenopus laevis oocyte... 28

F igu re 1.14 Proposal o f pseudoknotted tertiary structure for 5S rRNA from E. coli....29

F igu re 1.15 Three-dimensional stereo models of Xenopus laevis oocyte 5S rRNA...31

F ig u re 1.16 C om plex, nonidentical regions o f 5S rRNA required for different f u n c tio n s ... 33

F igure 2.1 The secondary structure o f Xenopus 5S rRNA with designations o f the sites o f protection by TFIIIA determined with hydioxyl radical... 38

F igure 2.2 Model for the interaction o f individual zinc fingers with 5S rRNA...40 F ig u re 2.3 Sketch o f the structure o f the Zif268-DNA com plex as determ ined by c ry s ta llo g ra p y ...4 1

(11)

XI F ig u re 2.4 Summary o f the base contacts made by the Zif268 zinc finger domain. The DNA is represented in a cylindrical projection... 42 F igure 2.5 Schematic representation '•f c,s model for binding o f TFIIIA to the 5S rRNA g en e I C R ... ... 45 F ig u re 2.6 Construction o f TFIIIA finger swap m utants...49 F ig u re 2.7 A) Schematic representation o f the oilogonucleotides used in the PCRs to generate the synthetic gene containing fingers 1-2, of TFIIIA and finger 3 of p43... 51 F ig u re 2.7 B) C onstruction o f Tp 3 m utant...52 F ig ure 2.8 Location o f scanning and point substitution mutations within the third finger o f T F II I A ... 55 F igure 2.9 Coomassie blue-stained 15% SDS-polyacrylamide gel of purified wild type and m utant TFIIIA s expressed in E. coli... 61 F ig ure 2.10 Nitrocellulose filter binding experiments with mutant forms of TFIIIAs...62 F ig u re 2.11 Susceptibility o f wild type and mutant forms o f TFIIIA to thermolysin d ig e s tio n ... 63 F ig u re 2.12 Structural representation o f a C2H2 zinc finger...68 F ig u re 2.13 Alignment of zinc finger sequences indicating the location o f amino acid resid u es th at c o n tact D N A ... 73 F ig u re 3.1 Finger comparison o f Xenopus laevis TFIIIA and p43... 78 F ig u re 3.2 (A). C onstruction o f pET-p43 plasm id... 81 F igu re 3.2 (B). Diagram of the pET-p43 construct used for the expression of p43 in E, c o li strain B L 2 1 (D E 3 )... 82

F ig u re 3.3 Coom assie blue-stained 15% SDS-polyacrylam ide gel o f purified p43 expressed in E. c o li... 86 F ig u re 3.4 Q uantitative binding o f p43 to nucleic acids.,... 88 F ig u re 3.5 A ctivity o f recom binant p43, and stoichiometry of the p43-5S rRNA c o m p le x ...,89

(12)

Xll Figure 3.6 C om petition binding assa y ... 92 Figure 3.7 Ka of the p43-5S rRNA interaction as a function o f M g^+ concentration...93 Figure 3.8 pH dependence o f the Ka s o f the p43-5S rRNA and TFIIIA-5S rRNA in te r a c tio n s ... ...9 4 Figure 3.9 KC1 concentration dependence of K as for the interaction o f p43 and TFIIIA w ith 5S rR N A ... 96 Figure 3.10 Temperature dependence o f the binding o f TFIIIA and p43 to 5S rRNA..97 Figure 3.11 M utant 5S rRNAs used to determine the recognition elements for p43 b in d in g ... 99 F igure 3 .1 2 N itro cellu lo se filter b inding assays fo r p43-5S rRN A m utant

in te r a c tio n s ... 100

Figure 3.13 Comparison of recognition elements for the binding o f TFIIIA and p43 to 5S rR N A ... 110 Figure 4.1 Alignment o f amino-terminal sequences in the ribosomal proteins homologous to ch ick en L 5 ... 114 F ig u re 4.2 The binding sites for E. coli ribosomal proteins L 5V L I 8 and L25 on 5S rRNA determ ined with a - s a r c in ... 116 Figure 4.3 C oom assie blue-stained 15% SD S-polyacrylam ide gel showing the purification of Xenopus laevis ribosomal protein L5 expressed in E. coli... 121 Figure 4.4 RNase footprinting o f L5-5S rRNA com plex... 125 Figure 4.5 RNase footprinting o f L5-5S rRNA mutant com plexes analyzed on 8% p o ly a c ry la m id e g e l... 126 Figure 4.6 RNase footprinting o f L5-5S rRNA mutant complexes analyzed on 12% p o ly a c ry la m id e g e l... 127 Figure 4.7 Summary o f the footprinting results o f L5-5S rR N A ...128 Figure 4.8 Summary o f the footprinting results o f the L5 protein with 5S rRNA mutant

(13)

xiii F igure 4.9 Summary of the footprinting results of the L5 pr it.un with 5S rRNA mutant 6 7 -7 0 ... 130

F igure 4.10 Summary of the footprinting results of the L5 protein with 5S rRNA mutant 7 3 -7 6 ... 131

F igu re 4.11 Comparison o f TFIIIA-5S rRNA (A), p43-5S rRNA (B) and L5-5S rRNA (C) fo o tp rin tin g re s u lts ... 137

(14)

xiv L IS T O F A B B R EV IA TIO N S

A: Adenine

ATP: Adenosine triphosphate BSA: Bovine serum albumin BPB: Bromophenol blue C: Cytosine

cDNA: Complementary deoxyribonucleic acid cpm : Counts per minute

CTP: Cytidine triphosphate

dATP: Deoxyadenosine triphosphate dCTP: Deoxycytidine triphosphate dG TP: Deoxyguanosine triphosphate dTTP: Deoxythymindine triphosphate DNA: Deoxyribonucleic acid

DTT: Dkhiothreitol E. coli: Eschericia coli

EDTA: Ethylenediamine-tetraacetic acid G: Guanine

GTP: Guanosine triphosphate ICR: Internal control region IE: Intermediate element

IP T G : Propyl-P-D-thiogalactopyranoside L5: Ribosomal protein L5

L18: Ribosomal protein L I 8 L25: Ribosomal protein L25 LB: Luria-Benton broth

(15)

X V mRNA: messenger ribonucleic acid

MW: Molecular weight N: Nucleotide

NMR: Nuclear magnetic resonance nt.: Nucleotide

PAGE: Polyacrylamide gel electrophoresis PEG : Ployethylene glycol

Pol. I ll: RNA polymerase III

PM SF: Phenylmethylsulfonyl Fluoride RNA: ribonucleic acid

rRNA: Ribosomal ribonucleic acid RNase: Ribonuclease

RN asin: Ribonuclease inhibitor RNP: Ribonucleoprotein particle S: Svedberg unit

SDS: Sodium dodecyl sulphate TBE: Tris; Borate; EDTA

TFIIIA : Transcription Factor IIIA TFIIIB : Transcription Factor IIIB T F IIIC : Transcription Factor IIIC itRNA: Transfer ribonucleic acid

Tris-H CI: Tris (hydroxymethyl) aminomethane-HCl U: Uracil

UTP: Uridine triphosphate W .G .: W heat germ W .T.: W ild-type XC: Xylene cyanol

(16)

xvi Xlo: Xenopus laevis oocyte

XIs: Xenopus laevis somatic Xlt: Xenopwi laevis oocyte trace

(17)

xvii A C K N O W L E D G M E N T

I thank my supervisory committee members, Dr. Juan Ausio, Dr. Edward E. Ishiguro, Dr. Nancy Sherwood, Dr. Trevor J. Trust for their valuable suggestions, and positive input over this period of study.

I am especially indebted to my supervisor, Dr. Paul J. Romaniuk, for his guidance, help, understanding and support-both intellectual and financial.

A special note of thanks is extended to my colleagues and friends, Kathy Bartilla, Jon Faris, Tanya Hemilton, Steve Hendy, Nik Veldhoen, Judith Wise for their help in routine laboratory work. The friendship has made my graduate training a pleasure.

Finally, special thanks are due my husband, Jian, for his understanding, support and love.

I am finacially supported by a University o f Victoria Graduate Fellowship and by a grant to Dr. Paul J. Romaniuk from Natural Sciences and Engineering Research Council of Canada.

(18)

CHAPTER 1 GENERAL INTRODUCTION

1.1. X E N O P U S T R A N S C R IP T IO N F A C T O R IIIA (T F IIIA )

1.1.1. Pu rification and Properties

Transcription factor IIIA (TFIIIA) was first purified to homogeneity from Xenopus oocytes using tedious conventional chromatography. Due to the abundance of TFIIIA in early developing X enopus oocytes, it was purified to homogeneity (Picard & Wegnez, 1979), even before it was recognized as a transcription factor (Engelke et al., 1980; Pelham & Brown, 1980). M ethods such as density-gradient centrifugation followed by DEAE cellulose chrom atography (Sm ith et al., 1984), or ion exchange chrom atography (Romaniuk, 1985) have later been developed to purify TFIIIA frorn Xenopus oocytes more efficiently. Active recombinant TFIIIA has been recently expressed in E. coli and purified to homogeneity yielding two to three milligrams o f protein from one liter o f bacterial culture (Del Rio & Setzer, 1991).

X enopus oocyte TFIIIA is a highly asymmetric single peptide o f 38.5 kD a in its

native state (Bieker & Roeder, 1984). This protein is required for the activation o f 5S rRNA gene transcription as well as the storage of 5S rRNA as a 7S ribonucleoprotein particle (RNP). It was shown that Xenopus TFIIIA may be involved in the unidirectional transport o f the 5S rRNA from the nucleus to the cytoplasm in the form of 7S RNPs. Yeast TFIIIA is slightly larger, about 50 kDa (Wang & Weil, 1989), while TFIIIA from Hela cells is smaller, about 35 kDa (Seifart et al., 1989). Human TFIIIA was purified and characterized to be about 40 kDa (Moorefield & Roeder, 1994). Despite the molecular w eight difference, these proteins are all specifically required for 5S rRNA gene transcription.

(19)

1.1.2. R egulation o f T FIIIA G ene Expression

TFIIIA is required along with TFIIIB, TFIIIC and RNA polymerase III, for transcription o f 5S rRNA genes in X enopus (Pelham & Brown, 1980). Changes in the cellular level of X enopus TFIIIA have been strongly implicated in the developmental control o f the 5S rRNA genes (Ginsberg e ta l., 1984; Honda & Roeder, 1980; Shastry et al., 1984), and therefore information on how TFIIIA is regulated is very helpful to our

understanding o f 5S rRNA gene regulation.

Although the altered levels o f TFIIIA gene expression during oogenesis and embryonic developmental o f Xenopus laevis have been observed for more than a decade (Honda & Roeder, 1980), the mechanism o f TFIIIA gene expression in different developmental stages is still not fully understood. Early immunological assays revealed that TFIIIA is not detectable in either mature oocytes or somatic cells (Honda & Roeder, 1980). Later immunological analysis of the levels of TFIIIA in oocytes and embryos indicated that the maximal steady state level of TFIIIA (about 1012 molecules/oocyte) is reached early in oogenesis, but drops dramatically in later stages (Shastry et al., 1984). Northern blot hybridization studies showed the level o f TFIIIA mRNA in a somatic cell to be approximately 106 times lower than in immature oocytes (Ginsberg et al., 1984; Taylor et al., 1986). This dramatic change in the TFIIIA mRNA content is mirrored by TFIIIA

protein levels, which drop from 1012 molecules per cell in early oogenesis to about 4 x 10^ molecules per cell in a tailbud stage embryo (Andrews & Brown, 1987), Thus, it appears that TFIIIA gene expression is developmentally controlled at the transcriptional level.

Regulation of gene expression is frequently achieved at the level o f transcription through the interaction o f specific proteins with elements located within the promoter region o f a gene. Ginsberg et al. (1984) first isolated and characterized a cDNA clone encoding TFIIIA protein. By examination of the cDNA sequence, they found a stretch of nucleotides that repeats twice near the 5 ’ end of the TFIIIA coding region and exhibits homology to the

(20)

“ B-box” o f the X eno pu s tm et gene promoter. Quantitation o f Southern hybridization signals suggested that the gene encoding TFIIIA is present at one copy per haploid genome (Ginsberg et al., 1984; Taylor & Brown, 1985). Tso et al. (Tso et al., 1986) isolated and sequenced the TFIIIA gene and flanking DNA from a genomic library. The TFIIIA gene in Xenopus laevis is about 11 kb in length and contains nine exons. In addition, a consensus

TATA box sequence, TATATAA, and a CAAT box sequence, GCCAATCC, are found at positions -32 and -96 respectively. The TFIIIA gene promoter structure is characteristic of other genes transcribed by RNA polymerase II. Positive and negative regulatory elements were identified in the promoter region o f Xenopus laevis TFIIIA gene (Scotto et al., 1989). 5 ’-delelion and internal deletion studies suggested that a negative elem ent lies between positions -306 and -289, as well as three positive-acting sequences located between positions -289 and -235, -250 and -173, and -144 and -101, respectively (Figure 1.1). The protein that binds to the positive element at position -271 to -253 was found to be related to the human adenovirus major late transcription factor (MLTF) by gel shift analysis (Scotto et al., 1989). Furthermore, this protein was shown to have DNA-binding properties similar to

M LTF by gel shift analysis, chemical footprinting, methylation interference, and point mutation analysis (Hall & Taylor, 1989). The MLTF-like protein-binding site in the TFIIIA gene promoter in cis stimulates transcription from the TFIIIA promoter in oocytes but not in s c ia tic cells. Therefore, this element may be oocyte-specific in the context o f the TFIIIA p a ter. However, both oocytes and somatic cells contain a MLTF-like protein that binds to the upstream cis element. The functional importance o f M LTF-like protein to the developmental regulation o f the TFIIIA gene is still not clear. Although a TFIIIA binding site on the 5 ’ flanking region o f its own gene was identified by a footprinting assay (Figure 1.1) (Hanas & Smith, 1990), there is so far no evidence for a role o f TFIIIA in the regulation of its own gene. More recently, Martinez et al. found that the Xenopus TFIIIA gene is not only transcribed by pol II in somatic cells but is also transcribed by pol III in these cells (Martinez et al., 1994). However, they did not demonstrate that the pol III

(21)

4 -3 2 6 -3 0 6 -2C9 - 2 5 3 -1 7 3 . 1 4 4 , 8 6 „7 8 , 3 2 , 4 + 1

t r}mb777frsz2s////A _ .__h s;;/A b A*

-250 -101 CAAT TATA -3 2 6 -2 6 4 T F I I I A binding region -271 -253 MLTF binding site

Figure 1.1 C is-acting elements in TFIIIA promoter. Shaded box represents negative dem ent. Striped boxes represents positive elements. Closed bar represents protein binding site.

(22)

transcripts were translated in these cells. It is still not clear what the biological role o f overlapping transcription by RNA pol II and pol III o f the X enopus TFIIIA gene is in somatic cells.

1.1.3. Structure o f TFIIIA

Xenopus TFIIIA contains nine C2H2 zinc fingers tandemly repeated through the N-

terminal tv/o thirds o f the protein. The proposed overall structure o f TFIIIA protein comes from the following lines o f evidence. First, zinc ions are involved in the binding o f the protein to both 5S rRNA and the 5S rRNA gene (Hanas et al., 1933: M iller et al., 1985). Analysis by atomic absorption spectroscopy revealed that purified I S RNP contained 7-11 zinc atoms per mole o f particle (Miller et al., 1985). The coordination o f each zinc ion with two sulphur and two nitrogen atoms was confirmed by extended X-ray absorption fine structure (EXAFS) (Diakun e ta l., 1986). Second, periodic intermediates and a limit-digf"t product o f 3 kDa o f TFIIIA were observed in proteolysis analysis (M iller et al., 1985). Third, the sequence o f TFIIIA derived from a cDNA clone revealed a 30 amino acid repeat unit containing two unique conserved cysteines and histidines (Figure 1.2) (Ginsberg et al., 1984; Brown et al., 1985). A schematic representation o f the proposed folding of a

TFIIIA zinc finger domain is shown in Figure 1.3. In this structure, two closely spaced cysteine and histidine residues coordinate with a single zinc atom and stabilize a ‘finger’ structure. This zinc finger m otif was later identified in the coding sequences o f many eukaroytic gene regulatory proteins (Berg, 1986; Gibson et al., 1988; Vincent, /986). TIk eukaroytic factors play an important role in cell growth and differentiation in a wide variety of organisms.

Am ino acid sequence analysis o f a variety o f zinc finger containing proteins revealed that each finger conforms to the general sequence repeat; (Tyr, Phe)-Xaa-Cys- Xaa2,4-Cys-Xaa3-Phe-Xaa5-Leu-Xaa2-Kis-Xaa3-5-His (Berg, 1990; Brown e ta l., 1985; Miller et al., 1985). Each repeat consists o f seven highly conserved amino acids including

(23)

6 I 0 13 IV 2 3 3 6 3 0 T G E K * P © V © . D G © D !\ R © T K K . . © K • I t © ‘ © 1 ( » G E K A L P V V y K R ) s II

C

_{- 0 l ©} _{S F A D © C A} _{A 0 N K N W K © 0} _{1 A ® l , f 1}

_K'CfO

_.'IV T C E K 1 P © P © K E E C © E K C 0 T S L II II © T • f i © S , X ' J © (17 T C E K ’ N © T © D S D G © D I, R © T T H A N M K ‘ K © , O J P ( H ) 91) N I K ! C V

(

y

)

V © H F E N © G K A © K K H N 0 © K • v f i O F ‘ S © 129 T 0 Q L • P ( ? ) E © P H E G © D *K R ( ? ) S L P S R © K » R © E K • V © 1 59 A G - - * - © P © K K D I) S © S @ V G K T W T © Y I. K © V A E C © 1 00 Q 0 - - ’ L A V © - - D V © N R K © R H K D Y © R • D © Q K * T © 2 1 4 E K E R T V © L © P R D G © D K S © T T A P N © R » S © [ 0 S F © 2-10 E E 0 R * P © V © E H A 0 © G K C © A M K K S © E ♦ R © S V < V © 2 7 G 2 7 7 ( D P E K R K L * K E K C P R P K R S L A S l l L T G Y I P P K S K C K 0 A O i l S V S C T E K T D S L V K N K P S G T E T | C S L V L D K L T I d ) 3-14

F igu re 1.2 Amino acid sequence of transcription factor IIIA from X. laevis oocyte. The fingers are numbered 1-9 on the left side of the diagram. (*) positions where an insertion sometimes occurs in the normal pattern. In the main body o f the repeats (-) indicates an alignment gap (after Miller et al., 1985).

(24)

F ig u re 1.3 Two-dim ensional folding scheme for a linear arrangem ent o f repeated domains ofTFIIIA (after Miller et al„ 1985).

(25)

two pairs o f cysteine and histidine residues that tetrahedrally coordinate a single zinc ion (Diakun e t al., 1986) and hydrophobic residues that form a hydrophobic con? within the zinc finger domain. Structural studies by NMR (Carr et al., 1990; Lee et al., 1989b; Michael et al., 1992; Parraga et al., 1988) and X-ray crystallography (Fairall et al., 1993; Pavletich & Pabo, 1991; Pavletich & Pabo, 1993) revealed that the C2H2 zinc fingers contain a two strand anti-parallel P-sheet followed by an a-helix (Figure 1.4). Each finger adopts a very similar tertiary conformation resulting in a highly compact globular nucleic acid binding domain. However, due to variations o f certain key amino acids, each zinc finger recognizes and binds to different DNA (or RNA) sequences (Pavletich & Pabo,

1991; Fairall eta l., 1993; Pavletich & Pabo, 1993).

1.1.4. Function o f TFIIIA

Initially, TFIIIA from cell extracts was found to be required for transcription o f 5S rRNA genes (Engelke et al., 1980). In vitro studies show that 5S rRNA synthesis is completely dependent on TFIIIA in combination with transcription factors IIIB and IIIC (Lassar et al., 1983). Transcriptional activation of 5S rRNA genes by TFIIIA is initiated by the binding o f TFIIIA to the internal control region (ICR) (from approximately +50 to +97) o f the 5S rRNA gene. Detailed information on TFIIIA-5S rRNA gene association is discussed in Chapter 2. The binding of TFIIIA to the 5S rRNA gene is followed by the binding o f TFIIIC, TFIIIB and RNA polym erase III (Bicker et al., 1985). These transcription factors in association with the 5S rRNA gene and RNA polymerase III form a stable transcription complex. The function o f TFIIIA in 5S rRNA gene transcription u to induce efficient binding of TFIIIC to the box A regions in the ICR of the 5S rRNA gene by either changing the conformation o f 5S rRNA gene in such a way as to make it more amenable to TFIIIC interaction (Engelke et al., 1980; Fiser-Littell & Hanas, 1988) or by interacting with TFIIIC through specific protein-protein interactions. The C-tcrminal domain o f TFIIIA was found to be required for transcriptional activation (Smith et al.,

(26)

9

F igure 1.4 Structure o f the Xfirt-31 zinc finger derived from NMR studies (after Lee et al., 1989b).

(27)

1984) and could m ediate interactions with TFIIIC. The result o f such protein-protein interaction would be the proper positioning o f TFIIIB by TFIIIC. Recently, a short region o f 14 to 18 amino acids within the C-term inal region o f TFIIIA was identified as an indispensable sequence for transcriptional activation (Mao & Darby, 1993).

In Xenopus oocytes TFIIIA acts as a positive transcription factor and also Interacts with 5S rRN A to form 7S ribonucleoprotein particles (RNP). This second activity of TFIIIA serves in the transport o f 5S rRNA from the nucleus and the storage of 5S rRNA within the cytoplasm until it is required for ribosomal assembly (Guddat e t al., 1990; Picard & W egnez, 1979). The studies on the interaction of TFIIIA and 5S rRNA is discussed in detail in Chapter 2.

1.2. 5S R IB O S O M A L RN A G E N E S O F X E N O P U S L A E V IS

1.2.1. T h e O rg a n iz a tio n o f 5S rR N A G enes

Xenopus laevis 5S rRNA genes are organized into three gene families: major oocyte

(Xlo) (Fedoroff & Brown, 1978), trace oocyte (Xlt), and somatic (Xls) 5S DNA (Peterson e t al., 1980). Each gene family is organized in clusters of simple tandem repeats. All three

gene types are transcribed in Xenopus oocytes, but only the somatic genes are active in Xenopus somatic cells (Ford & Southern, 1973; Wegnez et al., 1972).

There are 20,000 copies o f m ajor oocyte 5S rRNA genes per haploid Xenopus genome. Each copy is composed o f two distinct halves: a conserved GC-rich half that carries the gene and the pseudo-gene (M iller et al., 1978), and a more variable AT-rich half, the spacer DNA, that carries a number o f repeated AT-rich blocks (Fedoroff & Brown, 1978). The pseudo-geue has no known function. The AT-rich spacer varies in length from about 360 to 570 or more nucleotides. The GC-rich half of the repeating unit contains a single long duplication o f 174 nucleotides (Miller et al., 1978). The relative locations o f the gene and pseudo-gene in the repeating unit in Xenopus laevis 5S DNA are

(28)

indicated in Figure 1.5. DNA methylation experiments show that m ost o f the CpG dinucleoudes in 5S DNA are at least partially methylated. Clusters o f four or more consecutive T residues preceeded by a GC-rich region in the noncoding DNA strand acts as a transcriptional termination signal (Bogenhagen & Brown, 1981). The transcription products o f the X lo 5S rRNA gene include a 130-base-long transcript in addition to the standard 120-base-long 5S rRNA transcript (Bogenhagen et al., 1980). 3 ’ exonuclease activity degrades the pseudo-gene 5S rRNA transcript and processes the major oocyte 5S rRNA transcript in Xenopus oocytes (Xing & Worcel, 1989).

The somatic 5S rRNA genes (400 copies per haploid X en o p u s genome) are transcribed in both oocyte and somatic cells (Peterson et al., 1980). There are six nucleotide differences between Xlo and Xls 5S rRNA genes. The spacers are completely different except for short conserved elements near the 5 ’ and 3 ’ end o f the genes. The Xls 5S rRNA genes have a GC-rich spacer, homogenous repeat lengths and no “pseudo genes”.

The last family of 5S rRNA gene is the trace oocyte type. There are about 2,000 o f these 5S rRNA genes per haploid complement of DNA. The X lt 5S rRNA gene has some oocyte and some somatic-specific residues, as well as nucleotides that differ from both types o f 5S rRNA genes (Brown et al., 1977). Like the somatic 5S DNAs, the Xlt 5S DNA has a homogenous length repeat and unique nucleotide sequence in its spacer (Peterson et al., 1980). The spacer of Xlt 5S DNA has the AT-rich sequence characteristic o f the m ajor oocyte 5S DNAs, and contains one duplication that differs in sequence approximately 40%.

1.2.2. 5S rRNA G ene Prom oter

(29)

12 H i n d H H a e l l l H o e 1H H in d m. 3 6 0- 5 7 0 - 5 8- ■193 -H a e -HI •4 3 -A, A 2 5 -G E N E 3' PSEUDOGENE ■REGION A ( A T - r i c h s p a c e r ) R E G I O N B

F igu re 1.5 Organization o f the repeating unit in X. laevis 5S DNA. The repeating unit has been divided into an AT-rich (A) and a GC-rich (13) region (after Miller et al., 1978),

(30)

been studied extensively. A detailed analysis using deletion mutants within the flanking and coding sequences initially determined base pairs 50 to 83 (where 1 denotes the site o f transcription initiation) as required for transcription initiation (Bogenhagen et al., 1980; Sakonju et al., 1980). Studies using a series of point mutants reveal that the promoter extends from base pair 50 to 97 o f the 5S rRNA gene (Pieler e t al., 1985; Pieler et al.,

1985). The internal control region (ICR) o f the 5S rRNA gene consists o f three distinct sequences : box A (50 to 64), the intermediate element (67 to 72) and box C (79 to 97) (Pieler et al., 1987; You e ta l., 1991) (Figure 1.6). Removal o f the 5 ’-flanking sequence from 5S rRNA gene has a small effect on transcriptional efficiency (Reynolds & Peck, 1981). The box A sequence is also found in the internal control region of tRNA genes (Ciliberto et al., 1983), whereas the intermediate and box C elements are specific to 5S rRNA gene. Both 5S rRNA and tRNA genes belong to the RNA polym erase III gene families and share common transcription factors (TFIIIB and TFIIIC) (Lassar et al., 1983; Shastry, 1993).

The internal control regions of somatic and oocyte 5S rRNA genes are very similar, but not identical. The main difference is in the box A region. Oocyte 5S rRNA genes contain guanine, thymine and adenine in positions 53,55 and 56 respectively, whereas the somatic genes contain cytosine, adenine and guanine at the those positions. The other difference is a cytosine in position 79 o f the oocyte genes which is changed into thymine in the somatic genes.

Studies by in vitro transcription and the formation o f initiation complexes o f 5S rRNA gene revealed that box A has a relatively low affinity for TFIIIA and is directly involved in the binding o f TFIIIC (Keller et al., 1990). The intermediate element and box C are the main determinants of TFIIIA binding (Pieler et al., 1987; Sakonju & Brown, 1982; Vrana et al., 1988). Sequence changes in the spacer regions between these promoter elements do not influence transcription (Pieler et al., 1985).

(31)

14 64 67 ₉₇ +120

H

30 47 53 55 56 79 Xlo: T A G T A C Ms: C G C A G T

Prom oter region (ICR) » |

F ig u re 1.6 The structure o f Xenopus laevis 5S rRNA genes, A refers to the box A promoter element, IE refers to the intermediate element of the promoter, and C refers to the box C promoter element.

(32)

15 1.2.3. D ev e lo p m e n ta l R e g u la tio n o f S o m atic a n d O o cy te 5S rR N A G en e E x p re s s io n

Oocyte and somatic 5S rRNA genes o f Xenopus laevis differ slightly in sequence (Figure 1.6). A lthough both genes are transcribed by RNA polym erase III during oogenesis, after fertilization and development o f the embryo the oocyte-specific genes are repressed, and the somatic 5S rRNA genes remain active. Interestingly, both gene families have similar c/s-acting elements that are recognized by the same transcription factors but they are controlled differently. What is the mechanism o f the developmental switch that turns off oocyte 5S rRNA gene transcription? A number of hypotheses have been proposed to explain this regulation.

1.2.3.1. T r a n s c r ip tio n co m p lex es

W olffe and Brown proposed a model for the understanding o f the molecular mechanisms that establish and maintain the pattern of differential gene activity in 5S rRNA gene transcription (W olffe, 19^*4; W olffe & Brown, 1988). This model includes the transcription complex, chromatin assembly and DNA replication.

Transcription o f a SZ . r<NA gene requires three transcription factors: TFIIIA, TFIIIB and TFIIIC. TFIIIA \s a single peptide protein which binds to the ICR o f the 5S rRNA gene. In yeast and human, TFIIIC is a complex protein consisting of four or more polypeptide chains (Conesa et al., 1993; Kovelman & Roeder, 1992; Parsons & Weil, 1990). In yeast and large eukaryotes, TFIIIB is comprised of three or more polypeptides, including the TATA binding protein (TBP) (Hernandez, 1993; Kassavetis et al., 1991; Rigby, 1993). TFIIIB is the key general factor necessary for the pol III initiation process, v/hereas TFIIIA and TFIIIC function as assembly factors (Kassavetis et al., 1990). These three transcription factors interact with the 5S rRNA gene in a defined order. TFIIIC recognizes the 5S rRNA gene only after TFIIIA is bound. Then, TFIIIB binds to the

(33)

TFIIIA/TFIIIC/5S rRNA gene complex. TFIIIB does not recognize the 5S rRNA gene itself but relies on protein-protein contact with TFIIIA and/or TFIIIC bound to the gene (Kassavetis e ta l., 1990).

The binding o f the TFIHA/5S rRNA gene complex by TFIIIC appears to play a role in the developmental regulation o f 5S rRNA genes. TFIIIA alone interacts with oocyte and somatic 5S rRNA genes with identical affinities (M cConkey & Bogenhagen, 1988). Contacts between TFIIIC and box A o f the ICR (around +53, +55 and +56) leads TFIIIC to interact preferentially with the TFIIIA complex bound to the somatic rather than the oocyte 5S rRNA gene (Keller et al., 1992; Keller et al., 1990; Seidel & Peck, 1992; Wolffe & Brow n, 1988) (Figure 1.7). Seidel and Peck (1992) determ ined that differential transcription o f somatic and oocyte-type 5S rRNA genes is a consequence of vastly different rates o f stable complex assembly. Once the complex is formed, transcription complexes on both types o f gene are stable and are transcribed at nearly equivalent rates.

I.2 .3 .2 . C h ro m a tin assem b ly a n d DNA re p lic a tio n

In Xenopus somatic cells the somatic 5S rRNA genes are active and the oocyte 5S rRNA genes are repressed. A competition between transcription factors and core histones for stable association with the prom oter elements of 5S rRNA genes contributes significantly to the selective repression o f oocyte 5S r'RNA genes. In vitro and in vivo studies using plasmid DNAs indicate that differences of 50-fold in transcription o f oocyte and somatic genes might be attributed to this competition (Andrews & Brown, 1987; Bouvet et al., 1994; Brown & Schlissel, 1985). W hat mechanism allows transcription factors to gain access to genes under normal physiological conditions when the histones and other chromosomal proteins are also associated with DNA? The staged assembly of chromatin following replication and nucleosome positioning are the main answers to this question (Figure 1.8). Nucleosome assembly follows a defined order. First a tqtramcr of

(34)

17

Oocyte Gene Som atic Gene

Equivalent Association (+ T F IIIA ) Differential Association { + T F IIIC ) i ~ * i i N 1 ■ t i t t * & f l t — o e y v .

F igure 1.7 Differential transcription factor association with oocyte and somatic 5S rRNA genes influences transcription during development. After TFIIIA forms a specific complex (Kd = ~10‘9M) with both oocyte and somatic 5S rRNA genes, TFIIIC then recognizes TFIIIA bound to both 5S rRNA genes. However, because of sequence differences at one o f the key contacts made by TFIIIC (vertical arrows), it binds the TFIIIA/oocyte 5S rRNA gene complex better than TFIIIA-somatic 5S rRNA gene complex (after Wolffe, 1994).

(35)

2 o a D O

|

a 18

f:

x 'C i

s \ 5 t

‘Eco f i \ S tu. *

k

V

₊ O’) cr. O Mh DU a. 2 2 2 P o < P Occ £u X CO u z < p 2 § icc X o

/

1 '

_Vi:

_L

V

¥ £8 Zu.

It

t

ft

o ♦ _ ■$ <D 3 s i 1* N Z h I I < CQ O

F ig u re 1.8 Model for gene programm ing during chromatin assembly on a 5S rRNA gene. (A) A histone tetramer (open ellipsoid) binds to the region from nucleotide -50 to +70 o f the 5S rRNA gene. TFIIIA can recognize nucleotides +81 to +91 of the 5S rRNA gene. Addition o f TFIIIC and TFIIIB lead to displacement of the histone tetramer and assembly o f a transcription complex. (B) If two histone H2A/H2B dimers (hatched crescents) bind to the histone tetramer (H3/H4)2-5S rRNA gene complex, then TFIIIA cannot bind to 5S DNA and transcription is repressed. (C) Acetylated histone octamer bound to 5S DNA would not prevent TFIIIA binding. Addition of TFIIIC and TFIIIB is proposed to displace the entire acetylated histone octamer (after Wolffe, 1994).

(36)

histones (H3/H4) is deposited, followed by two dimers o f histones H2A/H2B and finally histone H I (Almouzni et al., 1990; Worcel et al., 1978). Upon binding o f the histone tetramer (H3/H4) to the 5S rRNA gene, the 3 ’-end of the ICR which is the key contact for TFIIIA binding remains exposed in the histone-DNA complex (Hayes & Wolffe, 1992). After histones H2A/H2B bind to the complex, the key base contacts o f the TFIIIA binding site are no longer accessible (Hayes & Wolffe, 1992; Lee e t al., 1993). However, it is still not clear if nucleosome positioning does have influence on the developmental regulation of the 5S rRNA genes, since nucleosomes were found to site on different sequences o f the Xlo, Xls and Xbs 5S rRNA genes (Gottesfeld & Bloomer, 1980; Gottesfeld, 1987; Lee et al., 1993).

The competition between TFIIIA and histones H2A/H2B for stable association with the key promoter element at the 3 ’end o f the ICR might determine whether a 5S rRNA gene will be transcriptionally active or not. If TFIIIA is in excess it will bind first and the gene will have the potential to be active (Figure 1.8A). If TFIIIC stabilizes TFIIIA binding to somatic 5S rRNA genes, histones H2A/H2B would not be able to repress transcription, and transcription proceeds. If histones H2A/H2B are in excess they will occupy the promoter before TFIIIA can bind, and the formation of the transcription complex will be prevented (Figure 1.8B). Although assembled chromatin will stably repress transcription (Chirk & Wolffe, 1991; Morse, 1989), DNA replication will displace the core histones and the competition between histones and transciption factors for association with the 5S rRNA gene will begin again (Wolffe, 1991).

Acetylation o f the N-terminal tails o f the core histones decreases their interaction with PN A and facilitates the binding o f TFIIIA with the 5S rRNA gene (Figure 1.8C; Lee et al., 1993). Histone H4 was found to be diacetylated immediately following synthesis

(Jackson et al., 1987). This diacetylated form o f histone H4 is stored within the Xenopus egg and is incorporated into embryonic chromatin (Dimitrov et al., 1993).

(37)

L inker histones, such as histone H I, are found to have an im portant role in differential transcription o f the 5S rRNA genes during X enopus em bryogenesis. The presence o f histone H I was found necessary in maintaining repression o f oocyte 5S rRNA genes in somatic cells (Schlissel & Brown, 1984). Normal somatic histone H I protein is not found in the Xenopus eggs (Dimitrov et al., 1993; Hock et al., 1993; Wolffe, 1989). Its mRNA is synthesized in the oocyte, but stored in a transcriptionally inactive ‘m asked’ form (Bouvet e ta l., 1994; Tafun & Wollfe, 1993). After fertilization somatic hLione HI protein starts to accumulate (Dimitrov et al., 1993; Wolffe, 1989). Reconstitution studies show that addition o f histone H I to mixtures of somatic and oocyte 5S rRNA genes selectively represses transcription o f oocyte 5S rRNA genes (Chipev & Wolffe, 1992; Wolffe, 1989). Results from in vitro studies reveal that the flanking sequences of Xenopus 5S rRNA genes determ ine differential inhibition of transcription by III histone

(Jerzmanowski & Cole, 1990). In vitro (Andrews & Brown, 1987; Wolffe, 1989) and in vivo (Bouvet et al., 1994) studies led to the conclusion that both TFIIIA limitation and

histone H I accumulation independently regulate differential 5S rRNA gene transcription (Figure 1.9). Variation in the amount o f histone HI in chromatin does not significantly influence somatic 5S rRNA gene transcription. However the incorporation o f histone III into chromatin during embryogenesis directs the specific repression o f the Xenopus oocyte 5S rRNA genes (Bouvet et al., 1994).

Several other models such as the DNA replication expression model (Gottesfeld & Bloomer, 1982; W onnington et al., 1982) and the 42 kDa TFIIIA repressor model (Blanco et al., 1989) have been proposed to explain developmental regulation o f 5S rRNA gene

expression. In the replication model, active somatic 5S rRNA genes replicated earlier in S- phase would sequester limiting key transcription factors, whereas oocyte 5S rRNA genes replicated late would not do so in the absence o f key transcription factors, However, studies using

Xenopus

egg extracts indicate that differential replication has no effect on oocyte and somatic 5S rRNA gene transcription (Wolffe, 1993). Moreover, repressed

(38)

21 Oocyte Gene

CD

C

D

,

^ 0

Lr

_LL

_ttt

+TFIIIA --- + H1

Som atic Gene

Lr

F igu re 1.9 M odel for differential oocyte and somatic 5S rRNA gene expression. Additional TFIIIA promotes oocyte transcription complex retention, whereas additional histone H I promotes oocyte transcription complex displacement through alterations to chromatin structure (after Wolffe, 1994).

(39)

oocyte 5S rRNA genes rem ain inactive in vivo even with a considerable excess of transcription factors (Andrews & Brown, 1987; Bouvet et al., 1994). Therefore, it is unlikely that replication expression is responsible for regulating differential 5S rRNA gene expression. The key results on which the TFIIIA repressor model was based have not been reproduced by others (Brown, 1991) and could not be confirm ed by G ottesfeld (Gottesfeld, 1993).

In summary, developm ental^ regulated expression of 5S rRNA genes in Xenopus is extremely complex and can be explained by changes in transcription factor and histone interactions with somatic and oocyte genes. Protein-protein interactions determine the stability o f transcriptional complexes. A decline in transcription factor abundance and histone H I accumulation can account for the developmental repression of the oocyte genes while the somatic genes remain in an active state.

1.3. 5S R IB O S O M A L RNA

1.3.1. 5S rR N A a n d R ibosom al C o m p o n en ts

Ribosomal 5S rRNA is an essential component o f the ribosome. Ribosomes are ribonucleoprotein particles consisting of a small and a large subunit. The 70S ribosomes of E. coli contain 50S and 30S subunits. The 50S subunit consists o f 23S rRNA, 5S rRNA

and 34 proteins; the 30S subunit is composed o f 16S RNA and 21 proteins (Traub & Nomura, 1968). Eukaryotic ribosomes are larger and have a sedimentation coefficient of 80S. The 60S large subunit consists o f three ribosomal RNAs (28S, 5.8 S and 5S rRNA) and proteins; the 40S small subunit is made up of 18S rRNA and proteins. The eukaryotic ribosome has more protein per unit RNA as well as per ribosome than the prokaryotic ribosome.

E. coli 5S rRNA has been localized by immunoclectron microscopy (IEM) on the

(40)

1983). Three E. coli 5S rRNA binding proteins L5, L18 and L25 were also found to lie on the central protuberance (Oakes et al., 1986). It was shown by different approaches that ribosomal proteins L5, L18 and L25 interact specifically with 5S rRNA in E. coli (Garrett & Noller, 1979; Huber & Wool, 1984; Spierer & Zimmerman, 1978). In the mammalian cells, 5S rRNA binds only to L5 protein. The 5S rRNA/L5 complex is a precursor to ribosom e assembly (Steitz et al., 1988). In yeast, 5S rRNA is associated wiin the L5 homologue, YL3 (Nazar, 1979).

1.3.2. S tr u c tu r e o f 5S rR N A

5S rRNA is an integral, functionally essential constituent of the prokaryotic and eukaryotic ribosome. 5S rRNA is a 120 nucleotide-long molecule. A comparison o f nucleotide sequences of 5S rRNA from over 100 different sources suggests that 5S rRNA has been highly conserved during the course of evolution (Dams et al., 1983; Luehrsen & Fox, 1981). Knowledge o f the secondary and tertiary structure o f 5S rRNA is important in understanding the structure and function of the ribosome. The common secondary structure o f all 5S rRNAs includes helices I to V, connected by loops A to E.

Fox and Woese first proposed the model o f a base-pairing prokaryotic 5S rRNA secondary structure (Figure 1.10) by comparative analysis o f primary structure (Fox & Woese, 1975). This secondary structure has since been confirmed by a wide variety of techniques including chem ical m odification (N oller & G arrett, 1979), ribonuclease digestion (Douthwaite & Garrett, 1981), and NMR studies (Gewirth e ta l., 1987; Kime & Moore, 1983; White et al., 1992; Zhang & Moore, 1989). A more recent model o f the secondary structure of 5S rRNA from E. coli proposed by White et al (1992) is shown in Figure 111. NMR results revealed that helix I contains a G-U pair and has unpaired bases at its termini (White et al., 1992). Spectroscopic data showed that the structure o f the loop E region o f the 5S rRNA from E. coli differs from the standard, phylogenetically derived

(41)

24 HO . U 6 C C U 0 9 C 9 0 t I I I I I I I U-ACOOACCGUC 9 C 0 0 U0 UOCCOC, A —’ 0 U. C - 0 ,c-C U G A I I I I ,0 A C U U I 0 'a-a. 0 -c

F ig u re 1.10 Schem atic representation o f 5S rRNA secondary structure. Normal base pairs are connected by vertical lines, whereas G-U pairs arc indicated by dots (after Fox & Woese, 1975).

(42)

25 •3* S’ I»u U -I A- U C— G G— C G— C 115 A — U s C— G C— G G— C i n U — G » C— G A C___ a p r " u c c c c a u g c g a g a g u a g g"TT — g " I I U G G as I G G U G U G A U G G U A r C C G A 15 M 75 70 C G AG - c « U— G

IV

v

_v

G

_{C— G M}

c

C— G G— U “ C— G A G A— U 25 C

II

G— C 55 U— A .G—C M A 1 aG — C 50 A— U C — G U— A 35 AC CC *5A c G C ^ « |, A C r U U 40

III

F igu re 1.11 Secondary structure of the 5S rRNA from E. coli (after White et al., 1992). RNA stem structures are identified by Roman numerals.

(43)

26 model for 5S rRNA, in which all base pairing is possible in loop E with the sequences aligned in parallel (Delihas et al., 1984).

For more than a decade Xenopus laevis oocyte 5S rRNA was used to study the structure o f eukaryotic 5S rRNA. Results of ribonuclease digestions (Andersen et al., 1984a) provided support for the generalized 5S rRNA secondary structure model derived from comparative sequence analysis (Figure 1.12) (Erdmann et al., 1984). In this model, loop E is a double stranded region susceptible to ribonuclease V I. Two bulged U residues at position 73 and 84 were confirmed by ribonuclease digestion (Andersen et al., 1984). Chemical probing o f loop E in 5S rRNA revealed that the loop acts like a helical region in which the bases are less reactive to chemical probes (Romaniuk et al., 1988; Westhof et al., 1989). Chemical reactivity analysis comfirmed that four bulged A residues at position 49, 50, 77 and 83, as well as a bulged C at position 63 ( Romaniuk et al, 1988). Recent NMR studies on the highly conserved loop E region of Xenopus laevis 5S rRNA indicated a guanosine residue at p osition 75 bulges into the major groove (Wimberly et al., 1993). The bulged U at position 73 was not confirmed by W imberly et al. (Wimberly et al.,

1993). The updated secondary structure o f 5S rRNA from Xenopus laevis oocyte is shown in Figure 1.13.

The universal model o f the secondary structure o f 5S rRNA first included non- W atson-Crick base-pairs for phylogenetic reasons. The best-characterized non-Watson- Crick base pairs are the G U wobble and G A pairs. Both occur in tRNA and have two hydrogen bonds (Saenger, 1984). Experimental evidence for the presence of A-G base pairs in nucleic acids came from ribonulease digestions in tRNA (Lockard & Kumar, 1981), and 16S rRNA (Douthwaite et al., 1983). Ribonuclease V I cleavages on 5S rRNA from X enopus laevis support the existence of A G base pairs in helices IV and V (Andersen e ta l., 1984). Furthermore, recent NMR studies identified the highly conserved G99 A77 base pairing (Wimberly et al., 1993).

(44)

27 V U U C G G A U G C U G 120 A* A 50 10- A C . 11 „ aAG DI * 5 S ? G - 9 C G C c c A C C C U G UG C C U G A U ^o\, ry ° y * ™ * Z A rCSOlCU G • C C A U A G I I l G • C n r A • U “ C • G TO C • G A • U - M U • A - u A • G 100 A • U G • A G • C G . C U Ue o Y C « G C * G G . A . C • G " C • G 90 A G G A ; u : c U A °

Figure 1.12 Proposed Xlo major oocyte 5S rRNA secondary structural model (after Andersen et al, 1984)

(45)

28 5 g c c u a c g g c 0 Ac c a c c c u g a A \ r r t U C U ° G jU U U C G G A U G c u g © • • • • • • • • ® ? p . U .G .A U © uc " U B Q G A c , C G G A C l ) m o U o g c « U A G A a C U « G « C U A A A U a g G » C 50 V A • U C * G 70 C « G A * U U ° U A o A G loo AoU GoA ’o ' G • C G » C U IJ *0 C * G C • G I A G « U IV C • G C • G “ A G ® G A

F ig u re 1.13 The secondary structure o f 5S rRNA from Xenopus laevis oocyte. Loop structures are identified by capital letters. Stem structures are identified by Roman numerals.

(46)

29

A

n

5'

3'

F igu re 1.14 Proposal o f pseudoknotted tertiary structure for 5S rRNA from E. coli. (A) Schematic model o f the structure. (B) Schematic base pairing o f the structure. Arrows point to the tertiary interactions that lead to pseudoknot formation (after Goringer & Wagner, 1988).

(47)

There is little information on the tertiary structure o f 5S rRNA. A pseudoknotted tertiaiy structural model for 5S rRNA from E, coli was proposed by Goringer & Wagner (Figure 1.14) (Goringer & Wagner, 1988). Several other tentative models for 5S rRNA tertiary structure described a folded structure with long-range interaction between loop C and either the internal loop E or external loop D (Hancock & Wagner, 1982; Nazar, 1.991; Pieler & Erdmann, 1982). In the higher order structural model proposed by Westhof et a l . (1989), 5S rRNA adopts a distorted Y-shape, in which a short stalk is made by stem 1 and the two arms o f the Y are made by stems 2 and 3 (Figure 1.15). This model does not account for any long-range tertiary interactions between loop C and loop D or region E. Structural studies using a rhodium probe (Chow et al., 1992) and site-directed mutagenesis (de Stevenson et al., 1991) provide further evidence for the absence of any long-range tertiary interactions between loop C and loop D or Tegion E o f Xenopus laevis oocyte 5S rRNA.

1.3.3. Function o f 5S rRNA

Ribosomal RNAs are critical for ribosomal architecture and function. In >itro studies reported that 5S rRNA was involved in the assembly o f biologically active 50S subunit o f B. stearothermophilus (Erdmann et al., 1971). Omission o f 5S rRNA prevents L5 protein from binding to the reconstituted particle (Horne & Erdmann, 1972). In vitro hybridization studies suggested that the base-pairing between 5S rRNA in the large ribosomal subunit and 18S (16S) rRNA in the small subunit might be involved in the reversible association of ribosomal subunits (Azad, 1979). Dohme and Nierhaus (Dohme & Nierhaus, 1976) found that reconstituted 50S subunits lacking 5S rRNA had a drastic reduction in peptidyltransferase activity. Photocrosslinking results indicated that stcm-loop IV o f 5S rRNA from E. coli lies close to the peptidyltransferase center (Dontsova et al.,

(48)

31

( a )

Figure 1.15 Three-dimensional stereo models o f Xenopus laevis oocyte 5S rRNA. (a) Continuous lines represent the phosphate backbone which has the Watson-Crick base-pairs whereas broken lines depict the phosphate backbone which has the non-canonical or tertiary base-pairs. (b) Atomic views in the same orientation. Heavy lines represent the phosphate backbone (after Westhof et al., 1989).

(49)

o f some aminoacyl-tRNA synthetases. GTPase and ATPase activity was also discovered in the E. coli 5S rRNA-L18-L25 complex (Gaunt-Klopfer & Erdmann, 1975).

The structural requirements o f 5S rRNA for nuclear transport, 7S RNP assembly and 60S ribosomal subunit assembly were elucidated through the analysis of the behavior o f wild type and mutant 5S rRNAs after microinjection into the cytoplasm o f Xenopus oocytes (Allison et al., 1993). Complex, nonidentical structural features within the central domain o f 5S rRNA are required for different functions (Figure 1.16).

1.3.4. T he B iological Pathway o f 5S rRNA in X e n o p u s Oocytes

Eukaryotic 5S rRNA is transcribed by RNA polymerase III. In Xenopus laevis, the 5S rRNA genes contain an internal promoter comprised o f about 50 base pairs. In addition to RNA polymerase III, multiple transcription factors are required for accurate and efficient transcription initiation on 5S rRNA genes. The transcription factors and the 5S rRNA gene assem ble into a transcription complex which serves as a substrate for transcription initiation by RNA polym erase III. This transcription complex remains functionally stable through many rounds of transcription. A cluster o f four or more T residues surrounded by GC-rich sequence at the end of 5S rRNA genes acts as the signal to terminate transcription.

The typical pathway o f 5S rRNA in somatic cells, such as Ilela cells, involves transport from the nucleus where it is synthesized by RNA polymerase III to the nucleolus for ribosome assembly. However, 5S rRNA in Xenopus oocytes takes a different journey o f nucleocytoplasmic transport. In Xenopus oocytes, ribosome assembly occurs late in oogenesis, but 5S rRNA is synthesized in large amounts before other components of the ribosome are available.Therefore, 5S rRNA is stored in 7S or 42S RNP storage particles for many days or weeks in the early oocytes. Microinjection experiments found that newly transcribed nuclear 5S rRNA transiently interacts with a 50-kDa La protein (Guddat et al,,

(50)

33 I G C C U A C G G • • • • • • • o • U U U C G G A U G C U G A A G . A U III 120 ₁₁₀ h“ f e ( T l A f c C C U G f — (a ) • • p b m E ® . . . _ _ " L _ G G IU G G G A C l. C G G E H1

K f —S

* A

u

A G s a 'a ' G » C A , " ' G C — C U G A • • • • • G A C U C • C • U • G to A G G i A G c A iooJa {D g gV c G y » c U V U 80 ' C > G C * G I A G * U C * G C # G A G G A O'. U C , i £ L U A U c T j 40 V s s V IV 90

_®

Key n u c le a r im port

CZ3

TFIIIA binding

~ * rib o so m e inco rp o ratio n

F igure 1.16 Complex, nonidentical regions o f 5S rRNA required for different functions, (after Allison eta l., 1993)

(51)

1990). The La protein is then replaced by either ribosomal protein L5 o r the 5S rRNA gene-specific TFIHA. Each o f these two RNPs migrates out of the nucleus to accumulate in the cytoplasm. However, in immature oocytes TFIIIA is in vast excess over L5, hence TFIIIA plays the m ajor role in nuclear export. Allison et al. (1991) found that during oogenesis 5S rRNA migrates from the nucleus to the cytoplasm for storage in the 7S RNP with TFIIIA. The cytoplasmically stored 5S rRNA associates with L5 during vitcllogenic stages. The L5/5S rRNA complex is then translocated to the nucleus and hence to the nucleoli fo r ribosome assembly. When ribosomes are treated with EDTA a 5S rRNA-L5 complex (5S RNP) is released (Blobel, 1971). 5S rRNA from Xenopus oocyte is shuttled between the nuclear and cytoplasmic compartments of the oocyte during development in a complicated pathway involving different protein associations.

Tw o kinds o f 5S rRNA-storage particles were found in previtellogenic Xenopus oocyte. The 7S RNP contains a single molecule each o f 5S rRNA and TFIIIA (Pelham & Brown, 1980), while the 42S RNP consists of a 50 kD protein, a 43 kD protein, tRNA and 5S rRNA in the m olar ratio 2:1:3:1 (Picard et al., 1980). The 50 kD protein interacts with the tRNA molecule and the 43 kD protein interacts with 5S rRNA. Previtellogenic oocytes o f Xenopus laevis are known to contain very large amount of tRNA and 5S rRNA (Denis & Mairy, 1972; Ford, 1971). These RNA species make up about 80% o f total oocyte RNA. Only a very small proportion o f 5S rRNA is associated with the ribosomes in these cells (Denis & Mairy, 1972; Ford, 1971). About half the 5S rRNA is associated with tRNA in 42S RN P (Ford, 1971; Denis & Mairy, 1972). The remainder o f 5S rRNA is associated with TFIIIA within 7S RNP (Picard & Wegnez, 1979). No 7S RNPs could be detected in somatic cells (Honda & Roeder, 1980), although TFIIIA binds both somatic and oocyte 5S rRNAs in vitro (Romaniuk et al., 1987). TFIIIA has dual functions involving both the storage o f 5S rRNA and acting as a positive transcription factor for expression of 5S rRNA genes (Honda & Roeder, 1980). P50, a 50 kDa protein in 42S RNP, was identified as a homologue of translation elongation factor l a (E F la ) (Mattaj et al„ 1987). P50 shares

Protein-nucleic acid interactions in Xenopus laevis Oocyte 5S Ribosomal RNA gene transcription

TABLE OF CONTENTS

CHAPTER 1

GENERAL INTRODUCTION

t r}*mb777frsz2s////A _ .__h s;;/A b A

C

K'CfO

(

)

H

|

f: