For cells to accurately read out the genomic content, high fidelity during transcription is required. This is mainly established by the accuracy of the active centre of RNA polymerase (RNAP). Based on in vitro experiments with Escherichia coli RNAP it was also suggested that proofreading of transcription via RNA hydrolysis by RNAP may contribute to overall fidelity and processivity. RNAP’s intrinsic cleavage activity is stimulated by the highly conserved Gre factors suggesting that Gre factors in this way further contribute to transcription fidelity. The in vivo role of Gre however, remains poorly understood. Furthermore, limited functional studies have been performed in bacteria outside E. coli. Here we examine the role of the Gre factor of the Gram-positive human pathogen Streptococcus pneumoniae, GreA. We show that GreA is not essential in S. pneumoniae although ΔgreA cells show a slower growth rate and morphological defects. To examine if transcription fidelity is affected by greA deletion in vivo, we constructed a plasmid that contains a constitutively expressed lacZ gene containing a stop codon mutation. Functional LacZ will thus be only produced if errors in transcription or translation are regularly made. Using this in vivo fidelity system we show that cells mutated for greA show significant levels of β-galactosidase activity, indicating that GreA promotes transcription fidelity in vivo. To explore the mechanism of GreA-dependent transcription fidelity in more detail, we have purified S. pneumoniae GreA and S. pneumoniae RNAP and performed in vitro transcription assays. We show that S. pneumoniae GreA indeed improves transcription fidelity in vitro. Interestingly, S. pneumoniae RNAP seems to be less processive than other bacterial RNAPs, forming strong species-specific pauses. S. pneumoniae GreA helps in overcoming these pauses and thereby improves RNAP’s processivity, thus increasing RNAP’s overall elongation rate. Genetic switches that depend on critical thresholds in concentration of regulator protein should be especially responsive to changes in transcription fidelity and rate. In line with this idea, the greA mutant was unable to become naturally competent for DNA uptake, a developmental process that is under the control of a sensitive genetic switch. Our results therefore suggest that fidelity/processivity of transcription facilitated by GreA is crucial for cell well being, and may play an important role in the control of genetic switches.
Created: 23rd Nov 2011 at 16:39
Last updated: 26th Aug 2014 at 14:20