Helix-loop-helix motif in HIV-1 Rev.
Circular dichroism (CD) spectra of C-terminal deletion mutants of the HIV-1 Rev protein, Rev M9 delta 14 (missing aa 68-112) and Rev M11 delta 14 (lacking aa 92-112), indicated that Rev contains 46-49 residues in alpha-helical conformation within the N-terminal 71 or 95 amino acids of the 116 residue protein. Complexation with a 40-nucleotide fragment of the Rev responsive element, RRE, (G39 to C78), containing the minimal element for Rev binding, induced an A to B form structural transition in the RRE fragment, whereas the percentage of alpha-helical conformation in the protein stays constant on substrate binding. When complexed to the RNA, neither mutant protein showed structural changes upon raising the temperature to 40 degrees C, as determined by the lack of decrease of the signal intensity at 222 nm, indicative for alpha-helical conformation. In contrast, Rev M9 delta 14, which is shorter than Rev M11 delta 14 by 24 amino acids, in the absence of RNA, lost about 60% of the spectral minima at 222 nm at the same temperature. The Rev M11 delta 14 mutant, in the absence of RNA, showed a decrease of 20% in spectral intensity upon heating to 40 degrees C. Free and RNA-bound mutant proteins showed reversible transitions upon heating to 80 degrees C and subsequent cooling down to 10 degrees C overnight. The Rev peptide Cys 75-93, spanning the Rev transactivation domain, showed secondary structure in 40% and 60% hexafluoropropanol (HFP) solutions.(ABSTRACT TRUNCATED AT 250 WORDS)
Auer
M
M
Sandoz Research Institute, Vienna, Austria.
Gremlich
H U
HU
Seifert
J M
JM
Daly
T J
TJ
Parslow
T G
TG
Casari
G
G
Gstach
H
H
D000595:Amino Acid Sequence; D001483:Base Sequence; D002942:Circular Dichroism; D015694:Gene Products, rev / Q000096:biosynthesis / Q000737:chemistry* / Q000378:metabolism; D015497:HIV-1 / Q000378:metabolism*; D018257:Helix-Loop-Helix Motifs; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D010446:Peptide Fragments / Q000138:chemical synthesis / Q000737:chemistry; D011487:Protein Conformation; D012313:RNA / Q000737:chemistry* / Q000378:metabolism; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism; D017384:Sequence Deletion; D015533:Transcriptional Activation; D054321:rev Gene Products, Human Immunodeficiency Virus
D015694:Gene Products, rev; D010446:Peptide Fragments; D011994:Recombinant Proteins; D054321:rev Gene Products, Human Immunodeficiency Virus; D012313:RNA