Alignment of the two domains of the hairpin ribozyme-substrate complex defined by interdomain photoaffinity crosslinking

被引:47
作者
Pinard, R [1 ]
Heckman, JE [1 ]
Burke, JM [1 ]
机构
[1] Univ Vermont, Dept Microbiol & Mol Genet, Burlington, VT 05405 USA
基金
美国国家卫生研究院; 英国医学研究理事会;
关键词
photoaffinity; crosslinking; azidophenacyl; hairpin; ribozyme;
D O I
10.1006/jmbi.1999.2626
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
The hairpin ribozyme-substrate complex contains two independently folding domains that interact with one another to form a catalytic complex. However, little is known about the key structural elements involved in these tertiary interactions. Here, we report the use of a photochemical crosslinking method to investigate the relative proximity and orientation of the two domains of the hairpin ribozyme. This method allows the incorporation of a photochemical azidophenacyl group at specified positions within synthetic oligoribonucleotides. Photocrosslinking was performed following the assembly of four RNA oligonucleotides into active ribozyme-substrate complexes. Two photoagent attachment sites in the substrate binding strand within domain A (between positions A7-G8 and A10-G11) and three in the 5' strand of domain B (A20-G21, A22-A23 and A24-C25) were studied. Several crosslinks between the substrate binding strand and the 5' segment of domain B were detected. All of the photoagent-specific crosslinked species were dependent upon proper assembly and folding of the ribozyme-substrate complex. In addition, a substrate base mutation (G(+1) to A(+1)) that prevents the docking of the two domains, blocks the crosslink formation. Four interdomain crosslinks (A7-G8/C25-A26 (two species); A10-G11/A22 and A24-C25/C12-G13) have been shown to retain catalytic activity. Taken together, these results indicate that the characterized crosslinks provide important information concerning the alignment of the two domains and accurately reflect the active docked conformation of the molecule. (C) 1999 Academic Press.
引用
收藏
页码:239 / 251
页数:13
相关论文
共 28 条
[11]   RNA CATALYTIC PROPERTIES OF THE MINIMUM (-)STRSV SEQUENCE [J].
HAMPEL, A ;
TRITZ, R .
BIOCHEMISTRY, 1989, 28 (12) :4929-4933
[12]   The solvent-protected core of the hairpin ribozyme-substrate complex [J].
Hampel, KJ ;
Walter, NG ;
Burke, JM .
BIOCHEMISTRY, 1998, 37 (42) :14672-14682
[13]   SEQUENCES REQUIRED FOR SELF-CATALYZED CLEAVAGE OF THE SATELLITE RNA OF TOBACCO RINGSPOT VIRUS [J].
HASELOFF, J ;
GERLACH, WL .
GENE, 1989, 82 (01) :43-52
[14]   Construction of hairpin ribozymes with a three-way junction [J].
Komatsu, Y ;
Shirai, M ;
Yamashita, S ;
Ohtsuka, E .
BIOORGANIC & MEDICINAL CHEMISTRY, 1997, 5 (06) :1063-1069
[15]   MODIFICATION OF PRIMARY STRUCTURES OF HAIRPIN RIBOZYMES FOR PROBING ACTIVE CONFORMATIONS [J].
KOMATSU, Y ;
KANZAKI, I ;
KOIZUMI, M ;
OHTSUKA, E .
JOURNAL OF MOLECULAR BIOLOGY, 1995, 252 (03) :296-304
[16]   Enhanced folding of hairpin ribozymes with replaced domains [J].
Komatsu, Y ;
Kanzaki, I ;
Ohtsuka, E .
BIOCHEMISTRY, 1996, 35 (30) :9815-9820
[17]   SEQUENCE-ANALYSIS OF 5'[P-32] LABELED MESSENGER-RNA AND TRANSFER-RNA USING POLYACRYLAMIDE-GEL ELECTROPHORESIS [J].
LOCKARD, RE ;
ALZNERDEWEERD, B ;
HECKMAN, JE ;
MACGEE, J ;
TABOR, MW ;
RAJBHANDARY, UL .
NUCLEIC ACIDS RESEARCH, 1978, 5 (01) :37-56
[18]   ACCEPTOR HELIX INTERACTIONS IN A CLASS-II TRANSFER-RNA SYNTHETASE - PHOTOAFFINITY CROSS-LINKING OF AN RNA MINIDUPLEX SUBSTRATE [J].
MUSIERFORSYTH, K ;
SCHIMMEL, P .
BIOCHEMISTRY, 1994, 33 (03) :773-779
[19]   CIRCULARLY PERMUTED TRANSFER-RNAS AS SPECIFIC PHOTOAFFINITY PROBES OF RIBONUCLEASE-P RNA STRUCTURE [J].
NOLAN, JM ;
BURKE, DH ;
PACE, NR .
SCIENCE, 1993, 261 (5122) :762-765
[20]   Base and sugar requirements for RNA cleavage of essential nucleoside residues in internal loop B of the hairpin ribozyme: Implications for secondary structure [J].
Schmidt, S ;
Beigelman, L ;
Karpeisky, A ;
Usman, N ;
Sorensen, US ;
Gait, MJ .
NUCLEIC ACIDS RESEARCH, 1996, 24 (04) :573-581