Probing the details of the HIV-1 Rev-Rev-responsive element interaction: effects of modified nucleotides on protein affinity and conformational changes during complex formation.
The solution structure of the human immunodeficiency virus type 1 (HIV-1) Rev-responsive element (RRE) has been investigated by enzymic and chemical structural probing of a 71 nt RRE transcript. The minimum sequence information required to maintain recognition by the Rev protein has previously been mapped to a 29 nt stem-loop structure, known as minSLIIB. The key recognition target is a single-stranded RNA bubble at the base of the RNA stem. The fine details of RNA recognition have been probed using chemically synthesized minSLIIBs containing variant base or sugar residues at sites within the bubble. These have been analysed by gel retardation assays and their relative affinities for Rev protein determined. Complex formation between the wild-type minSLIIB RRE and Rev protein was also monitored using CD spectroscopy, which suggests a change in RNA conformation upon Rev binding. The spectral change is consistent with localized melting of RNA, leading to a decrease in the level of base stacking and/or a change in base tilting, during formation of the complex. Deoxynucleotide substitutions on just one side, the 5' side, of the bubble inhibit the conformational change detected by CD. The data are consistent with a dynamic interaction between Rev and its target site. The contact points between Rev and the RRE were probed directly using photo-cross-linking with either ribo-5-bromouridine- or ribo-4-thiouridine-substituted minSLIIBs. The data are consistent with protein-RNA contacts at the bottom of the bubble.
Renwick
S B
SB
Department of Genetics, University of Leeds, U.K.
Critchley
A D
AD
Adams
C J
CJ
Kelly
S M
SM
Price
N C
NC
Stockley
P G
PG
D001483:Base Sequence; D002942:Circular Dichroism; D015694:Gene Products, rev / Q000737:chemistry*; D015497:HIV-1 / Q000737:chemistry* / Q000235:genetics; D006860:Hydrogen Bonding; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D010777:Photochemistry; D011485:Protein Binding; D012367:RNA, Viral / Q000737:chemistry*; D016601:RNA-Binding Proteins / Q000737:chemistry*; D013329:Structure-Activity Relationship; D054321:rev Gene Products, Human Immunodeficiency Virus
D015694:Gene Products, rev; D012367:RNA, Viral; D016601:RNA-Binding Proteins; D054321:rev Gene Products, Human Immunodeficiency Virus