The ribonuclease H domain from the HIV-1 reverse transcriptase protein. The four active-site carboxylate residues are shown in magenta. Two bound manganese ions are shown as purple spheres. A bound inhibitor molecule, beta-thujaplicinol, is shown in green.[1]
Although the RT structures from human, murine and avianretroviruses display different subunits, the relative sizes, orientation and connection of the DNA polymerase and RNase H domains are strikingly similar. The RNase H domain occupies ~25% of the RT protein C-terminal. The DNA polymerase domain occupies ~55% of the RT protein N-terminal.[5]
The RNase H domains of MMLV and HIV-1 RT enzymes are structural very similar to the Escherichia coli and Bacillus halodurans RNases H as well as to human RNaseH1.[6][7][8][9][10] In general, the folded structures of retroviral RNase H domains take the form of 5-stranded mixed beta sheets flanked by four alpha helices in an asymmetric distribution. A notable difference between the various RNase H proteins is the presence or absence of the C-helix (present in E. coli, MLV and human RNases H, absent in HIV-1, B. halodurans and ASLV RNases H), a positively charged alpha helix also referred to as the basic loop or protrusion.[10] It is believed to have a role in substrate binding.[10]
Function
During reverse transcription of the viral genomic RNA into cDNA, an RNA/DNA hybrid is created. The RNA strand is then hydrolyzed by the RNase H domain to enable synthesis of the second DNA strand by the DNA polymerase function of the RT enzyme.[5] In addition, retroviral virions package a single tRNA molecule that they use as a primer during reverse transcription of the viral genomic RNA.[11] The retroviral RNase H is needed to digest the tRNA molecule when it is no longer needed. These processes happen in a Mg2+ dependent fashion.[12][13]
Retroviral RNases H cleave their substrates through 3 different modes:
RNA 5'-end directed cleavage 13-19 nucleotides from the RNA end.
DNA 3'-end directed cleavage 15-20 nucleotides away from the primer terminus.
The two end-directed modes are unique to the retroviral RNases H because of a number of effects of the associated polymerase domain of retroviral RT.[6] In the more universal internal cleavage mode, the RNases H behave as typical endonucleases and cleave the RNA along the length of a DNA / RNA hybrid substrate in the absence of any ‘end’ effects.[14][15][16][17]
^Worthington, Von (1993). Worthington Enzyme Manual. Worthington. p. 280.
^Katayanagi K, Miyagawa M, Matsushima M, Ishikawa M, Kanaya S, Nakamura H, Ikehara M, Matsuzaki T, Morikawa K (February 1992). "Structural details of ribonuclease H from Escherichia coli as refined to an atomic resolution". Journal of Molecular Biology. 223 (4): 1029–52. doi:10.1016/0022-2836(92)90260-q. PMID1311386.
^Katayanagi K, Miyagawa M, Matsushima M, Ishikawa M, Kanaya S, Ikehara M, Matsuzaki T, Morikawa K (September 1990). "Three-dimensional structure of ribonuclease H from E. coli". Nature. 347 (6290): 306–9. Bibcode:1990Natur.347..306K. doi:10.1038/347306a0. PMID1698262. S2CID4234320.
^Yang W, Hendrickson WA, Crouch RJ, Satow Y (September 1990). "Structure of ribonuclease H phased at 2 A resolution by MAD analysis of the selenomethionyl protein". Science. 249 (4975): 1398–405. doi:10.1126/science.2169648. PMID2169648.
^Taylor JM (March 1977). "An analysis of the role of tRNA species as primers for the transcription into DNA of RNA tumor virus genomes". Biochimica et Biophysica Acta (BBA) - Reviews on Cancer. 473 (1): 57–71. doi:10.1016/0304-419x(77)90007-5. PMID66067.