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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+OCEM+CA)
CARAC'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#ailaility unrestricted
Astract
All functional R*As contain 1ost-transcri1tionally modified
nucleotides "8-O-ri3osemethylation 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
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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 su3seuently 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$1
com1le5 are reuired for efficient catalysis
.inally, a hi2h-resolution crystal structure of C3f$-*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 structuraldifferences 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
