Isolation and characterization of transcription fidelity mutants

Abstract

Accurate transcription is an essential step in maintaining genetic information. Error-prone transcription has been proposed to contribute to cancer, aging, adaptive mutagenesis, and mutagenic evolution of retroviruses and retrotransposons. The mechanisms controlling transcription fidelity and the biological consequences of transcription errors are poorly understood. Because of the transient nature of mRNAs and the lack of reliable experimental systems, the identification and characterization of defects that increase transcription errors have been particularly challenging. In this review we describe novel genetic screens for the isolation of fidelity mutants in both Saccharomyces cerevisiae and Escherichia coli RNA polymerases. We obtained and characterized two distinct classes of mutants altering NTP misincorporation and transcription slippage both in vivo and in vitro. Our study not only validates the genetic schemes for the isolation of RNA polymerase mutants that alter fidelity, but also sheds light on the mechanism of transcription accuracy. This article is part of a Special Issue entitled: Chromatin in time and space.

Fig. 1.

Schematic illustration of a set of reporters used for the isolation/screening of RNA polymerase slippage mutants. (A) The yeast malE-trp1 reporter driven by the HIS3 promoter and with a spacer containing runs of A [A_(n)] is shown. In a (+1) out-of-frame construct, wild type (WT) formed only small-sized colonies on Synthetic Complete plates lacking tryptophan; however, Pol II slippage mutants appeared as larger-sized colonies. (B) The E. coli chromosomal lacI-lacZ reporter driven by a mutant Ptac promoter (Ptac*) and with a slippery spacer A_(n) is shown. In a 9A(−1) contruct, WT formed white color colonies on MacLac indicator plates after 15 h incubation and red color after more than 24 h. Slippage-enhancing (↑) RNAP mutants were red color after shorter periods of incubation while slippage-reducing (↓) RNAP mutants maintained white color even after a prolonged incubation.

Fig. 2.

Mapping of the amino acid residues involved in control of slippage in the structure of TEC by Pol II and bacterial RNAP. On the top: Structure of the RNA and DNA stands in Pol II TEC (from PDB: 2E2H, [19]). Three amino acid residues involved in control of slippage are highlighted in red (in Rpb1 and Rpb2 subunits). Template DNA/front-end DNA duplex (gray), RNA (green) and the incoming NTP in the active center (yellow) are shown. The rest of the structure is omitted for simplicity. The inset shows a view along the long axis of the RNA-DNA hybrid in TEC with the 3’ end of the RNA marked. On the bottom: The structure of TEC by T. thermpohilis RNAP (from PDB: 2O5J, [24]). The corresponding residues in the β subunit of E. coli RNAP carrying slippage mutations are shown in red. The other elements of TEC are color-coded the same as in the Pol II structure.

Fig. 3.

Test for slippage in vitro. (A) The cartoon shows the assembled TEC by yeast Pol II before incubation with 10μM ATP and 100μM labeled CTP. The horizontal arrow denotes the DNA sequence for halting of TEC due to the lack of GTP. The two cleavage CMP sites for RNase A (red) in the nascent RNA are shown. The different pattern of the digested RNA products was expected to accumulate depending on the slippage directionality. The bottom panel displays the actual data for wild type Pol II and the Rpb1-N488D mutant exhibiting the increase of the deletions and insertions in the 11A-tract. The corresponding length of the A-tract in the RNA is shown on the right side of the gel. Note, that digestion with RNase A allowed an unambiguous identification of the slippage products (lanes 2 and 4), which was impossible in the non-digested samples (lanes 1 and 3) because Pol II partially transcribed across the stop site due to the cross-contamination of the commercial stocks of ATP and CTP with residual amount of GTP. (B) Yeast Pol II and EcRNAP have the opposite slippage directionality in the 11A-tract under the identical transcription conditions.

Fig. 4.

Schematic illustration of the Cre-mediated capture of transcription errors. The 8A(+1) malE-cre reporter is out-of-frame, generating nonfunctional Cre recombinase. Transcriptional slippage can restore the reading-frame of Cre. The active Cre in turn removes neo^R from the second reporter ade6-AIlox::neo^R, generating active ADE6-AI. In an ade2 background, an ade6-AIlox::neo^R colony is white and ADE6-AI are red colonies. Red sectors, representing transcription slippage errors that produce active Cre, are more frequent in transcription slippage mutants. SD (splicing donor site) and SA (splicing acceptor site).

Fig. 5.

The rpb1-E1103G mutation highlights the role of the trigger loop in Pol II fidelity. Glu1103 residue of the trigger loop and Rpb9 subunit play a role in stabilization of the opened state of the Pol II active site, and attenuate sequestration of substrate NTP leading to the improved fidelity. The closed (shaded pink) and opened (red) states of the trigger loop are superimposed from the crystal structures of the elongation complex by the yeast Pol II [PDB: 2E2H and 1Y1V, respectively [19,25]]. Glu1103 and Thr1095 interact in the opened conformation of the trigger loop, but this interaction is disrupted by the loop closure on the NTP (yellow). Rpb9 subunit of Pol II (gold) forms a putative interaction with the opened trigger loop. All other elements are colored the same as in Fig. 2.