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Metal-responsive transcription factor 1 (MTF-1) specifically binds to metal response elements (MREs) associated with a number of metal- and stress-responsive genes. Human MTF-1 contains a cysteine-rich cluster, -632Cys-Gln-Cys-Gln-Cys-Ala-Cys638-, conserved from pufferfish to humans far removed from the MRE-binding zinc finger domain and just C-terminal to a previously mapped serine/threonine-rich transcriptional activation domain. MTF-1 proteins containing two Cys→Ala substitutions (C632A/C634A) or a deletion in this region altogether (Δ(632-644)) are significantly impaired in their ability to induce Zn(II)- and Cd(II)-responsive transcription of a MRE-linked reporter gene in transiently transfected mouse dko7 (MTF-1-/-) cells in culture under moderate metal stress but retain the ability to drive basal levels of transcription in a MRE-dependent manner in vivo and in vitro. In addition, the mutated proteins respond to induction by Zn(II) or Cd(II) with nuclear translocation and MRE binding activities comparable with wild-type MTF-1. Attempts to rescue the Δ(632-644) deletion mutant phenotype by insetting similar Cys-rich sequences from Drosophila MTF-1 were unsuccessful, suggesting that the structure of this motif within intact human MTF-1, rather than the simple presence of multiple closely spaced Cys residues, is required for function. This cysteine cluster therefore functions at a step subsequent to nuclear translocation and MRE-binding DNA to naked promoter-containing DNA and appears to be specifically required for MTF-1 to activate transcription in the presence of inducing heavy metal ions. [ABSTRACT FROM AUTHOR]

The role of the small RNA polymerase II subunit Rpb9 in transcriptional proofreading was assessed in vitro. Transcription elongation complexes in which the 3' end of the RNA is not complementary to the DNA template have a dramatically reduced rate of elongation, which provides a fidelity checkpoint at which the error can be removed. The efficiency of such proofreading depends on competing rates of error propagation (extending the RNA chain without removing the error) and error excision, a process that is facilitated by TFIIS. In the absence of Rpb9, the rate of error propagation is increased by 2- to 3-fold in numerous sequence contexts, compromising the efficiency of proofreading. In addition, the rate and extent of TFIIS-mediated error excision is also significantly compromised in the absence of Rpb9. In at least some sequence contexts, Rpb9 appears to enhance TFIIS-mediated error excision by facilitating efficient formation of a conformation necessary for RNA cleavage. If a transcription error is propagated by addition of a nucleotide to the mismatched 3' end, then the rate of further elongation increases but remains much slower than that of a complex with a fully base-paired RNA, which provides a second potential fidelity checkpoint. The absence of Rpb9 also affects both error propagation and TFIIS-mediated error excision at this potential checkpoint in a manner that compromises transcriptional fidelity. In contrast, no effects of Rpb9 on NTP selectivity were observed.