GreA and GreB proteins revive backtracked RNA polymerase in vivo by promoting transcript trimming

Abstract

The GreA and GreB proteins of Escherichia coli show a multitude of effects on transcription elongation in vitro, yet their physiological functions are poorly understood. Here, we investigated whether and how these factors influence lateral oscillations of RNA polymerase (RNAP) in vivo, observed at a protein readblock. When RNAP is stalled within an (ATC/TAG)(n) sequence, it appears to oscillate between an upstream and a downstream position on the template, 3 bp apart, with concomitant trimming of the transcript 3' terminus and its re-synthesis. Using a set of mutant E.coli strains, we show that the presence of GreA or GreB in the cell is essential to induce this trimming. We show further that in contrast to a ternary complex that is stabilized at the downstream position, the oscillating complex relies heavily on the GreA/GreB-induced 'cleavage-and-restart' process to become catalytically competent. Clearly, by promoting transcript shortening and re-alignment of the catalytic register, the Gre factors function in vivo to rescue RNAP from being arrested at template positions where the lateral stability of the ternary complex is impaired.

Fig. 1. Schematic models of a stable and an oscillating elongation complex halted in vivo by the lac repressor bound to its operator. The middle section of the figure shows the relevant part of plasmids pATC6a and pATC6b with the non-template strand sequence of the repeats. These plasmids, which have been described previously (Toulmé et al., 1999), are pKK232-8 derivatives that carry the β-lactamase (Amp) and chloramphenicol acetyltransferase (CAT) genes. The transcription of the cat gene initiated at the constitutive hisR promoter goes through the repeats and the operator sequences that are inserted at position +40 relative to the transcription start site. Positions –6 and –9 relative to the upstream edge of the operator motif are also indicated, as the mRNA start site (right-angled arrow). See text for details.

Fig. 2. Nuclease S1 mapping of the 3′ ends of RNAs produced from pATC6a in wild-type (Wt), greA^–, greB^– or greA^– greB^– (Dble) mutant strains. (A) Autoradiogram showing the distribution of the RNA 3′ ends. In this and subsequent figures, the + or – signs at the top of the lanes denote the addition or omission, respectively, of the inducer (IPTG). The arrowheads indicate the –6 and –9 positions of the 3′ ends of the transcript relative to the upstream edge of the operator motif. (B) Densitometric scans of the S1-protected bands obtained in the absence of IPTG, quantified and processed on a PhosphorImager (Molecular Dynamics) with Imagequant software Version 3.3 for data processing. The data reported in the graph point to variations in the intensity of the –6 and –9 signals in the greA^–, greB^– and greA^– greB^– mutant strains compared with the wild-type strain.

Fig. 3. In situ DMS modification patterns of the template strand of pATC6a in wild-type (Wt), greA^–, greB^– and greA^– greB^– (Dble) mutant strains. The position of the G-6 residue is indicated by an arrow.

Fig. 4. Primer extension analysis of CAA modification on the non-template strand of pATC6a in wild-type (Wt) or greA^– greB^– (Dble) mutant strains. The two arrows at positions –6 and –18 delimit the apparent footprint of the complex, which is in equilibrium between the upstream and downstream conformations.

Fig. 5. cat mRNA quantitation in wild-type (Wt), greA^–, greB^– and greA^– greB^– (Dble) mutant cells transformed with either pATC6a or pATC6b. (A) Autoradiogram of the extension products obtained after in vitro reverse transcription of the RNAs with ³²P primers hybridizing to either the cat or the bla transcripts. The asterisk indicates a non-specific site of arrest during reverse transcription of the cat mRNA. (B) Quantification of the extension products shown in (A) was performed and processed based on densitometric scans as described in Figure 2. For each lane, the value plotted indicates the relative level of cat mRNA synthesis, calculated as explained in the text. The error bars reflect the standard deviation from a mean of three independent experiments. The 1.0 value for the wild type is an arbitrary unit. On the right-hand side of the histogram, the data obtained with the pATC6b plasmid introduced in the wild-type and the double-mutant strains is also reported to underline the Gre factor-independent readthrough when the ternary complex is stabilized in the downstream position (experimental data not shown).