Tight Post-transcriptional Regulation of RecBCD Expression is Required for Optimal DNA Repair in Escherichia coli

Core Concepts

Hfq, an RNA-binding protein, post-transcriptionally regulates the expression of RecB, a key component of the RecBCD enzyme complex, to maintain its levels within an optimal range for efficient DNA double-strand break repair in Escherichia coli.

Abstract

The content describes the regulation of RecBCD expression, a crucial enzyme complex for DNA double-strand break repair in Escherichia coli. Key insights: RecB mRNA is present at very low levels and has a short lifetime, suggesting weak and constitutive transcription. However, RecB protein levels are tightly controlled, with lower cell-to-cell variability than expected. The RNA-binding protein Hfq post-transcriptionally regulates RecB expression. Hfq binds to the recB mRNA and represses its translation, but does not affect mRNA stability. In the absence of Hfq, RecB protein levels increase without a corresponding change in mRNA levels, indicating enhanced translation efficiency. Deleting the main Hfq-binding site in the recB mRNA 5'UTR also leads to increased RecB translational efficiency, confirming the specificity of Hfq regulation. Upon DNA double-strand break induction, RecB translational efficiency increases, potentially due to changes in Hfq availability, suggesting a broader role for post-transcriptional regulation in the DNA damage response. The Hfq-mediated post-transcriptional control helps maintain RecB protein levels within an optimal range, likely to avoid the detrimental effects of RecBCD overexpression on DNA repair. This study provides evidence that Hfq contributes to suppressing fluctuations in RecB protein copy numbers, highlighting the importance of post-transcriptional regulation in controlling noise in the expression of low-abundance essential enzymes.

Stats

RecB mRNA has a degradation rate of 0.62 min^-1, corresponding to a lifetime of 1.6 min. The average RecB protein concentration is 2.12 mol/μm^2 in wild-type cells and 2.28 mol/μm^2 upon DNA damage induction. The average number of RecB proteins produced per mRNA increases from 7.9 in wild-type to 19.7 upon DNA damage induction.

Quotes

"RecBCD has been shown to process the chromosome for up to ∼ 100 kb at ∼ 1.6 kb/s in live bacteria [5]. Such a potent DNA degradation activity is controlled by chi recognition [11]. However, because this recognition is probabilistic [12], RecBCD can degrade large chromosome fragments before DNA repair is initiated." "Whilst the biochemical and in vivo activities of RecBCD have been extensively studied ([11, 16, 17, 18, 19], and [7, 20] for review), less is known about the regulation of its expression."

Key Insights Distilled From

An Hfq-dependent post-transcriptional mechanism fine tunes RecB expression in Escherichia coli

by Kalita,I., I... at www.biorxiv.org 10-24-2021

https://www.biorxiv.org/content/10.1101/2021.10.23.465540v2

Deeper Inquiries

How might the Hfq-mediated post-transcriptional regulation of RecB expression be integrated with other stress response pathways in E. coli

The Hfq-mediated post-transcriptional regulation of RecB expression in E. coli can be integrated with other stress response pathways through a network of interactions and feedback mechanisms. Hfq is known to regulate a wide range of mRNAs, including those involved in stress responses and adaptation to environmental changes. By controlling the translation of RecB, Hfq can coordinate the expression of this essential DNA repair protein with other cellular processes in response to stress conditions. For example, Hfq may interact with sRNAs that are activated in response to DNA damage, leading to changes in the translation efficiency of RecB. This coordinated regulation ensures that the cell can efficiently repair DNA damage while maintaining overall cellular homeostasis. Additionally, Hfq has been linked to the DNA damage and SOS responses, suggesting that it plays a role in integrating RecB expression with the broader stress response network in E. coli.

What other DNA repair proteins or pathways might be subject to similar post-transcriptional control mechanisms involving RNA-binding proteins

Several other DNA repair proteins or pathways in E. coli may be subject to similar post-transcriptional control mechanisms involving RNA-binding proteins. For example, proteins involved in base excision repair, nucleotide excision repair, and mismatch repair pathways could be regulated by RNA-binding proteins like Hfq. These proteins are essential for maintaining genome integrity and responding to DNA damage, making them prime candidates for post-transcriptional regulation. Additionally, proteins involved in homologous recombination, such as RecA, could also be regulated at the post-transcriptional level to ensure proper DNA repair. By fine-tuning the expression of these DNA repair proteins, RNA-binding proteins can help cells respond effectively to DNA damage and maintain genomic stability.

Could the principles of post-transcriptional noise suppression observed for RecB expression be applied to engineer more robust gene expression systems in synthetic biology

The principles of post-transcriptional noise suppression observed for RecB expression could be applied to engineer more robust gene expression systems in synthetic biology. By understanding how RNA-binding proteins like Hfq regulate gene expression and reduce protein fluctuations, researchers can design synthetic circuits that exhibit more predictable and stable gene expression patterns. For example, incorporating Hfq-like regulators into synthetic gene networks could help minimize noise and variability in protein expression, leading to more reliable and consistent output. This approach could be particularly useful in applications where precise control of gene expression is critical, such as in metabolic engineering, biotechnology, and gene therapy. By leveraging the insights from natural regulatory mechanisms, synthetic biologists can create more efficient and reliable genetic circuits for a wide range of applications.