Structural And Biochemical Characterization Of Rna-Guided Modification Enzymes

Title page for ETD etd-11212005-141641

Type of Document Dissertation
Author Rashid, Rumana
Author's Email Address shaimee@yahoocom
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Title S'R(C'(RA) A*D B+OC,EM+CA)
C,ARAC'ER+-A'+O* O. R*A-G(+DED MOD+.+CA'+O*
E*-/MES
Degree 0hD
Department Chemistry and Biochemistry, De1artment of
Ad#isory $ommittee Ad#isor "ame Title
'imothy M )o2an Committee Chair
)loyd M E1stein Committee Mem3er
Michael S Cha1man Committee Mem3er
Ro3ert , Ree4es Committee Mem3er
%ey&ords R*A
Modifications
R*0
Bo5 C6D
Bo5 ,6ACA
Date of Defense "##$-!!-!7
A#aila'ility unrestricted
A'stract
All functional R*As contain 1ost-transcri1tionally modified nucleotides "8-O-ri3ose
methylation and 1seudouridylation are the t9o ma:or ty1es of modifications, 9hich occur 3y
3o5 C6D and 3o5 ,6ACA ri3onucleo1rotein ;R*0< com1le5es, res1ecti4ely Generally, the
2uide R*A 1art 3inds to the com1lementary re2ions in the tar2et R*As 3ut the actual
catalysis is carried out 3y the 1rotein 1art of the com1le5 +n eu=aryotes, these R*0s are
called small nucleolar ;sno<R*0 com1le5es and in Archaea these are =no9n as small
;s<R*0s 'he archaeal 3o5 C6D sR*0 is a com1le5 of three 1roteins> fi3rillarin, *o1$1 and
)7Ae ?hereas, 3o5 ,6ACA sR*0 contains four 1roteins> C3f$, *o1!#, Gar! and )7Ae
Durin2 the last t9o decades si2nificant 1ro2ress has 3een achie4ed in identifyin2 the
com1onents of these t9o classes of sR*0s 3ut less is =no9n a3out their mode of assem3ly,
ste1s of assem3ly and catalysis 1rocesses 'he 9or= of this manuscri1t descri3es the 3asic
mechanisms 2o4ernin2 1rotein-1rotein, 1rotein-R*A interactions 9ithin archaeal sR*0
1articles 3y em1loyin2 3iochemical and crystallo2ra1hic a11roaches
Durin2 the initial in4esti2ation, 2el mo3ility shift assay 9as used to study A ful2idus 3o5 C6D
R*0 com1le5 +t 9as esta3lished that )7Ae nucleates the 9hole com1le5 3y
chan2in2 the R*A conformation and su3se@uently 3indin2 9ith fi3rillarin6*o1$1 com1le5
9here *o1$1 9as o3ser4ed to 3e the R*A 3indin2 1rotein *e5t, 9e used 2el shift assay,
analytical ultracentrifu2ation, and in 4itro methylation assay to study the assem3ly 1rocess of
C6D R*0 com1le5 Our results su22ested that 9hile a 3o5 C6D sR*0 is ca1a3le of
asymmetric assem3ly, the symmetries in 3oth the 3o5 C6D R*A and in the fi3rillarin6*o1$1
com1le5 are re@uired for efficient catalysis
.inally, a hi2h-resolution crystal structure of C3f$-*o1!#-Gar! 1rotein com1le5 9as o3tained
from 0 furiosus 'he structure ca1tures a functional assem3ly state of the ,6ACA sR*0
1article and thus 1ro4ides a mechanistic understandin2 on ho9 this system 9or=s C3f$ shares
ma:or structural domains 9ith other 1seudouridine synthases, 3ut dis1lays structural
differences consistent 9ith its distinct function in R*A-2uided 1seudouridylation +n addition,
9e descri3ed the 1re4iously un=no9n structures of 3oth *o1!# and Gar!, and their essential
roles in 1seudouridylation 1rocess