Jawdekar Gauri W, Hanzlowsky Andrej, Hovde Stacy L, Jelencic Blanka, Feig Michael, Geiger James H, Henry R William
Department of Microbiology & Molecular Genetics, Michigan State University, East Lansing, Michigan 48824, USA.
J Biol Chem. 2006 Oct 13;281(41):31050-60. doi: 10.1074/jbc.M603810200. Epub 2006 Aug 9.
Human small nuclear RNA gene transcription by RNA polymerases II and III depends upon promoter recognition by the SNAPC general transcription factor. DNA binding by SNAPC involves direct DNA contacts by the SNAP190 subunit in cooperation with SNAP50 and SNAP43. The data presented herein shows that SNAP50 plays an important role in DNA binding by SNAPC through its zinc finger domain. The SNAP50 zinc finger domain contains 15 cysteine and histidine residues configured in two potential zinc coordination arrangements. Individual alanine substitution of each cysteine and histidine residue demonstrated that eight sites are important for DNA binding by SNAPC. However, metal binding studies revealed that SNAPC contains a single zinc atom indicating that only one coordination site functions as a zinc finger. Of the eight residues critical for DNA binding, four cysteine residues were also essential for both U1 and U6 transcription by RNA polymerase II and III, respectively. Surprisingly, the remaining four residues, although critical for U1 transcription could support partial U6 transcription. DNA binding studies showed that defects in DNA binding by SNAPC alone could be suppressed through cooperative DNA binding with another member of the RNA polymerase III general transcription machinery, TFIIIB. These results suggest that these eight cysteine and histidine residues perform different functions during DNA binding with those residues involved in zinc coordination likely performing a dominant role in domain stabilization and the others involved in DNA binding. These data further define the unorthodox SNAP50 zinc finger region as an evolutionarily conserved DNA binding domain.
RNA聚合酶II和III对人类小核RNA基因的转录取决于SNAPC通用转录因子对启动子的识别。SNAPC与DNA的结合涉及SNAP190亚基与SNAP50和SNAP43协同作用直接与DNA接触。本文给出的数据表明，SNAP50通过其锌指结构域在SNAPC与DNA的结合中发挥重要作用。SNAP50锌指结构域包含15个半胱氨酸和组氨酸残基，以两种潜在的锌配位排列方式构成。对每个半胱氨酸和组氨酸残基进行丙氨酸替换表明，有8个位点对SNAPC与DNA的结合很重要。然而，金属结合研究表明，SNAPC含有单个锌原子，这表明只有一个配位位点起锌指的作用。在对DNA结合至关重要的8个残基中，4个半胱氨酸残基分别对RNA聚合酶II和III转录U1和U6也至关重要。令人惊讶的是，其余4个残基虽然对U1转录至关重要，但可以支持部分U6转录。DNA结合研究表明，仅SNAPC与DNA结合的缺陷可以通过与RNA聚合酶III通用转录机制的另一个成员TFIIIB协同DNA结合来抑制。这些结果表明，这8个半胱氨酸和组氨酸残基在与DNA结合过程中发挥不同功能，其中参与锌配位的残基可能在结构域稳定中起主导作用，而其他参与DNA结合的残基则起不同作用。这些数据进一步将非传统的SNAP50锌指区域定义为一个进化上保守的DNA结合结构域。
