Secondary structure and mutational analysis of the ribosomal frameshift signal of Rous sarcoma virus

被引:40
作者
Marczinke, B [1 ]
Fisher, R [1 ]
Vidakovic, M [1 ]
Bloys, AJ [1 ]
Brierley, I [1 ]
机构
[1] Univ Cambridge, Dept Pathol, Div Virol, Cambridge CB2 1QP, England
基金
英国惠康基金; 英国医学研究理事会;
关键词
Rous sarcoma virus; retrovirus; frameshifting; RNA structure probing; pseudoknot;
D O I
10.1006/jmbi.1998.2186
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
Expression of the Gag-Pol polyprotein of Rous sarcoma virus (RSV) requires a -1 ribosomal frameshifting event at the overlap region of the gag and pol open reading frames. The signal for frameshifting is composed of two essential mRNA elements; a slippery sequence (AAAUUUA) where the ribosome changes reading frame, and a stimulatory RNA structure located immediately downstream. This RNA is predicted to be a complex stem-loop but may also form an RNA pseudoknot. We have investigated the structure of the RSV frameshift signal by a combination of enzymatic and chemical structure probing and site-directed mutagenesis. The stimulatory RNA is indeed a complex stem-loop with a long stable stem and two additional stem-loops contained as substructures within the main loop region. The substructures are not however required for frameshifting. Evidence for an additional interaction between a stretch of nucleotides in the main loop and a region downstream to generate an RNA pseudoknot was obtained from an analysis-of the frameshifting properties of RSV mutants translated in the rabbit reticulocyte lysate in vitro translation system. Mutations that disrupted the predicted pseudoknot-forming sequences reduced frameshifting but when the mutations were combined and should re-form the pseudoknot, frameshifting was restored to a level approaching that of the wild-type construct. It was also observed that the predicted pseudoknot-forming regions had reduced sensitivity to cleavage by the single-stranded probe imidazole. Overall, however, the structure probing data indicate that the pseudoknot interaction is weak and may form transiently. In comparison to other characterised RNA structures present at viral frameshift signals, the RSV stimulator falls into a novel group. It cannot be considered to be a simple hairpin-loop yet it is distinct from other well characterised frameshift-inducing RNA pseudoknots in that the overall contribution of the RSV pseudoknot to frameshifting is less dramatic. (C) 1998 Academic Press.
引用
收藏
页码:205 / 225
页数:21
相关论文
共 50 条
[41]   Mutational analysis of the RNA pseudoknot involved in efficient ribosomal frameshifting in simian retrovirus-1 [J].
Sung, D ;
Kang, H .
NUCLEIC ACIDS RESEARCH, 1998, 26 (06) :1369-1372
[42]  
TEN DE, 1995, RNA, V1, P146
[43]  
TEN DE, 1994, NUCLEIC ACIDS RES, V22, P2304
[44]  
TEN DE, 1992, BIOCHEMISTRY-US, V31, P11665
[45]   SEQUENCE RELATIONSHIPS OF TYPE-D RETROVIRUSES WHICH CAUSE SIMIAN ACQUIRED-IMMUNODEFICIENCY-SYNDROME [J].
THAYER, RM ;
POWER, MD ;
BRYANT, ML ;
GARDNER, MB ;
BARR, PJ ;
LUCIW, PA .
VIROLOGY, 1987, 157 (02) :317-329
[46]   TEMPERATURE-DEPENDENT CHEMICAL AND ENZYMATIC PROBING OF THE TRANSFER RNA-LIKE STRUCTURE OF TYMV RNA [J].
VANBELKUM, A ;
VERLAAN, P ;
KUN, JB ;
PLEIJ, C ;
BOSCH, L .
NUCLEIC ACIDS RESEARCH, 1988, 16 (05) :1931-1950
[47]   CLEAVAGE OF TRANSFER-RNA WITH IMIDAZOLE AND SPERMINE IMIDAZOLE CONSTRUCTS - A NEW APPROACH FOR PROBING RNA STRUCTURE [J].
VLASSOV, VV ;
ZUBER, G ;
FELDEN, B ;
BEHR, JP ;
GIEGE, R .
NUCLEIC ACIDS RESEARCH, 1995, 23 (16) :3161-3167
[48]   RNA PSEUDOKNOTS - STABILITY AND LOOP SIZE REQUIREMENTS [J].
WYATT, JR ;
PUGLISI, JD ;
TINOCO, I .
JOURNAL OF MOLECULAR BIOLOGY, 1990, 214 (02) :455-470
[49]   IMPROVED M13 PHAGE CLONING VECTORS AND HOST STRAINS - NUCLEOTIDE-SEQUENCES OF THE M13MP18 AND PUC19 VECTORS [J].
YANISCHPERRON, C ;
VIEIRA, J ;
MESSING, J .
GENE, 1985, 33 (01) :103-119
[50]  
YOUNG JF, 1983, ORIGIN PANDEMIC INFL, P129