Primer Grip As noted in the dsDNA section, the fingers, thumb, and palm subdomains act together as a kind of "molecular clamp," to position the dsDNA into the active site. This forms the "primer grip." It involves the b12-b13 hairpin loop of the 3 stranded, antiparallel b-sheets, b12-b13-b14 in the palm subdomain. It is composed of Trp229, Met230, Gly231 and Tyr232. It is believed that these residues are directly involved in the exact positioning of the terminal nucleotide 3'-OH group of the primer strand into the active site. Show me! In mutagenesis experiments, it has been observed that hydrophobic residues at positions 229, 232 and 234 are required for activity with a strong preference for Trp, Tyr, and Leu, respectively, giving further credence to the necessity of these residues in enzyme activity.1 The YMDD RT Active Site Motif Of the 139 contacts between the RT and the dsDNA , only two of these involve direct nucleotide base contacts: Tyr183and Met184! Both Tyr183 and Met184 are part of the conserved YMDD (i.e. tyrosine, methionine, aspartic acid, aspartic acid) active site motif that is highly conserved in retroviral RTs. Both Tyr183 and Met184 are the only ones that form close contacts (d<=3.8 Angstroms) with the dsDNA in the crystal structure (in vivo distances may differ due to physiological conditions). Tyr183 forms dual H-bonds with the cytosine oxygen (O2), in red, of the primer strand at a distance of 3.5 A and an Oh-H---O2 angle of 130o and the guanine nitrogen (N2), in blue, of the template strand at a distance of 3.7 A and an Oh-H---N2 angle of 151o. Show me! Replacement of Tyr183 with phenylalanine substantially reduces the transcription rates of HIV RT-1 by 20-30% from the wild-type.2 Further active site interactions involve the remaining aspartic acid residues in the YMDD motif include: Asp185, Asp186. Also, Asp110 is also believed to play an integral role due to its proximity to the active site. Show me! The Template Grip Motif Amino acid residues Asp76, Glu89, Gln151, Gly152, Lys154 and Pro157 have close contacts with the template strand. These comprise the template grip motif and are believed to be directly involved in positioning the template strand near the active site. Show me! 1 Xiong, Y., Eickbush, T. H., EMBO J., 9, 3353-3362, 1990; and Ding, J., et al., Structure, 3, 365-379. 2 See Boyer, P. et al., J. Virol., 66, 1031-1039, 1992; and Chao, S.-F. et al., Nucl. Acids Res., 23, 803-810, 1995.
In mutagenesis experiments, it has been observed that hydrophobic residues at positions 229, 232 and 234 are required for activity with a strong preference for Trp, Tyr, and Leu, respectively, giving further credence to the necessity of these residues in enzyme activity.1
The YMDD RT Active Site Motif Of the 139 contacts between the RT and the dsDNA , only two of these involve direct nucleotide base contacts: Tyr183and Met184! Both Tyr183 and Met184 are part of the conserved YMDD (i.e. tyrosine, methionine, aspartic acid, aspartic acid) active site motif that is highly conserved in retroviral RTs. Both Tyr183 and Met184 are the only ones that form close contacts (d<=3.8 Angstroms) with the dsDNA in the crystal structure (in vivo distances may differ due to physiological conditions).
Tyr183 forms dual H-bonds with the cytosine oxygen (O2), in red, of the primer strand at a distance of 3.5 A and an Oh-H---O2 angle of 130o and the guanine nitrogen (N2), in blue, of the template strand at a distance of 3.7 A and an Oh-H---N2 angle of 151o. Show me! Replacement of Tyr183 with phenylalanine substantially reduces the transcription rates of HIV RT-1 by 20-30% from the wild-type.2 Further active site interactions involve the remaining aspartic acid residues in the YMDD motif include: Asp185, Asp186. Also, Asp110 is also believed to play an integral role due to its proximity to the active site. Show me! The Template Grip Motif Amino acid residues Asp76, Glu89, Gln151, Gly152, Lys154 and Pro157 have close contacts with the template strand. These comprise the template grip motif and are believed to be directly involved in positioning the template strand near the active site. Show me! 1 Xiong, Y., Eickbush, T. H., EMBO J., 9, 3353-3362, 1990; and Ding, J., et al., Structure, 3, 365-379. 2 See Boyer, P. et al., J. Virol., 66, 1031-1039, 1992; and Chao, S.-F. et al., Nucl. Acids Res., 23, 803-810, 1995.
Replacement of Tyr183 with phenylalanine substantially reduces the transcription rates of HIV RT-1 by 20-30% from the wild-type.2
Further active site interactions involve the remaining aspartic acid residues in the YMDD motif include: Asp185, Asp186. Also, Asp110 is also believed to play an integral role due to its proximity to the active site. Show me! The Template Grip Motif Amino acid residues Asp76, Glu89, Gln151, Gly152, Lys154 and Pro157 have close contacts with the template strand. These comprise the template grip motif and are believed to be directly involved in positioning the template strand near the active site. Show me! 1 Xiong, Y., Eickbush, T. H., EMBO J., 9, 3353-3362, 1990; and Ding, J., et al., Structure, 3, 365-379. 2 See Boyer, P. et al., J. Virol., 66, 1031-1039, 1992; and Chao, S.-F. et al., Nucl. Acids Res., 23, 803-810, 1995.
The Template Grip Motif Amino acid residues Asp76, Glu89, Gln151, Gly152, Lys154 and Pro157 have close contacts with the template strand. These comprise the template grip motif and are believed to be directly involved in positioning the template strand near the active site. Show me! 1 Xiong, Y., Eickbush, T. H., EMBO J., 9, 3353-3362, 1990; and Ding, J., et al., Structure, 3, 365-379. 2 See Boyer, P. et al., J. Virol., 66, 1031-1039, 1992; and Chao, S.-F. et al., Nucl. Acids Res., 23, 803-810, 1995.
1 Xiong, Y., Eickbush, T. H., EMBO J., 9, 3353-3362, 1990; and Ding, J., et al., Structure, 3, 365-379.
2 See Boyer, P. et al., J. Virol., 66, 1031-1039, 1992; and Chao, S.-F. et al., Nucl. Acids Res., 23, 803-810, 1995.