Role of the trigger loop in translesion RNA synthesis by bacterial RNA polymerase.

DNA lesions can severely compromise transcription and block RNA synthesis by RNA polymerase (RNAP), leading to subsequent recruitment of DNA repair factors to the stalled transcription complex. Recent structural studies have uncovered molecular interactions of several DNA lesions within the transcription elongation complex. However, little is known about the role of key elements of the RNAP active site in translesion transcription. Here, using recombinantly expressed proteins, in vitro transcription, kinetic analyses, and in vivo cell viability assays, we report that point amino acid substitutions in the trigger loop, a flexible element of the active site involved in nucleotide addition, can stimulate translesion RNA synthesis by Escherichia coli RNAP without altering the fidelity of nucleotide incorporation. We show that these substitutions also decrease transcriptional pausing and strongly affect the nucleotide addition cycle of RNAP by increasing the rate of nucleotide addition but also decreasing the rate of translocation. The secondary channel factors DksA and GreA modulated translesion transcription by RNAP, depending on changes in the trigger loop structure. We observed that although the mutant RNAPs stimulate translesion synthesis, their expression is toxic in vivo, especially under stress conditions. We conclude that the efficiency of translesion transcription can be significantly modulated by mutations affecting the conformational dynamics of the active site of RNAP, with potential effects on cellular stress responses and survival.

Distinct effects of DNA lesions on RNA synthesis by Escherichia coli RNA polymerase.

DNA lesions can severely compromise genome stability and lead to cell death if unrepaired. RNA polymerase (RNAP) is known to serve as a sensor of DNA damage and to attract DNA repair factors to the damaged template sites. Here, we systematically investigated the ability of Escherichia coli RNAP to transcribe DNA templates containing various types of DNA lesions, and analyzed their effects on transcription fidelity. We showed that transcription is strongly inhibited on templates containing cyclobutane thymine dimers, 1,N6-ethenoadenine and abasic sites, while 8-oxoguanine and thymine glycol have mild effects on transcription efficiency. Similarly to many polymerases, E. coli RNAP follows the "A" rule during nucleotide insertion opposite abasic sites and bulky lesions, and can also incorporate and efficiently extend an adenine nucleotide opposite 8-oxoguanine. Mutations in RNAP regions around the templating nucleotide decrease the efficiency of translesion synthesis, likely by altering the RNAP-template contacts in the active site. Thus, DNA lesions can lead to distinct outcomes in transcription, depending on the severity of the damage and contacts of the damaged template with the active site of RNAP.

The active site residues Gln55 and Arg73 play a key role in DNA damage bypass by S. cerevisiae Pol η.

Eukaryotic DNA polymerase eta (Pol η) plays a key role in the efficient and accurate DNA translesion synthesis (TLS) opposite UV-induced thymine dimers. Pol η is also involved in bypass of many other DNA lesions but possesses low fidelity on undamaged DNA templates. To better understand the mechanism of DNA synthesis by Pol η we investigated substitutions of evolutionary conserved active site residues Gln55 and Arg73 in Saccharomyces cerevisiae Pol η. We analyzed the efficiency and fidelity of DNA synthesis by the mutant Pol η variants opposite thymine dimers, abasic site, thymine glycol, 8-oxoguanine and on undamaged DNA. Substitutions Q55A and R73A decreased the catalytic activity and significantly affected DNA damage bypass by Pol η. In particular, the Q55A substitution reduced the efficiency of thymine dimers bypass, R73A had a stronger effect on the TLS-activity opposite abasic site, while both substitutions impaired replication opposite thymine glycol. Importantly, the R73A substitution also increased the fidelity of Pol η. Altogether, these results reveal a key role of residues Gln55 and Arg73 in DNA synthesis opposite various types of DNA lesions and highlight the evolutionary importance of the Pol η TLS function at the cost of DNA replication accuracy.

Roles of the active site residues and metal cofactors in noncanonical base-pairing during catalysis by human DNA polymerase iota.

Human DNA polymerase iota (Pol ι) is a Y-family polymerase that can bypass various DNA lesions but possesses very low fidelity of DNA synthesis in vitro. Structural analysis of Pol ι revealed a narrow active site that promotes noncanonical base-pairing during catalysis. To better understand the structure-function relationships in the active site of Pol ι we investigated substitutions of individual amino acid residues in its fingers domain that contact either the templating or the incoming nucleotide. Two of the substitutions, Y39A and Q59A, significantly decreased the catalytic activity but improved the fidelity of Pol ι. Surprisingly, in the presence of Mn(2+) ions, the wild-type and mutant Pol ι variants efficiently incorporated nucleotides opposite template purines containing modifications that disrupted either Hoogsteen or Watson-Crick base-pairing, suggesting that Pol ι may use various types of interactions during nucleotide addition. In contrast, in Mg(2+) reactions, wild-type Pol ι was dependent on Hoogsteen base-pairing, the Y39A mutant was essentially inactive, and the Q59A mutant promoted Watson-Crick interactions with template purines. The results suggest that Pol ι utilizes distinct mechanisms of nucleotide incorporation depending on the metal cofactor and reveal important roles of specific residues from the fingers domain in base-pairing and catalysis.

Distinct functions of regions 1.1 and 1.2 of RNA polymerase σ subunits from Escherichia coli and Thermus aquaticus in transcription initiation.

RNA polymerase (RNAP) from thermophilic Thermus aquaticus is characterized by higher temperature of promoter opening, lower promoter complex stability, and higher promoter escape efficiency than RNAP from mesophilic Escherichia coli. We demonstrate that these differences are in part explained by differences in the structures of the N-terminal regions 1.1 and 1.2 of the E. coli σ(70) and T. aquaticus σ(A) subunits. In particular, region 1.1 and, to a lesser extent, region 1.2 of the E. coli σ(70) subunit determine higher promoter complex stability of E. coli RNAP. On the other hand, nonconserved amino acid substitutions in region 1.2, but not region 1.1, contribute to the differences in promoter opening between E. coli and T. aquaticus RNAPs, likely through affecting the σ subunit contacts with DNA nucleotides downstream of the -10 element. At the same time, substitutions in σ regions 1.1 and 1.2 do not affect promoter escape by E. coli and T. aquaticus RNAPs. Thus, evolutionary substitutions in various regions of the σ subunit modulate different steps of the open promoter complex formation pathway, with regions 1.1 and 1.2 affecting promoter complex stability and region 1.2 involved in DNA melting during initiation.