Regulatory RNAs in bacteria

Abstract

Bacteria possess numerous and diverse means of gene regulation using RNA molecules, including mRNA leaders that affect expression in cis, small RNAs that bind to proteins or base pair with target RNAs, and CRISPR RNAs that inhibit the uptake of foreign DNA. Although examples of RNA regulators have been known for decades in bacteria, we are only now coming to a full appreciation of their importance and prevalence. Here, we review the known mechanisms and roles of regulatory RNAs, highlight emerging themes, and discuss remaining questions.

Figure 1. Gene Arrangement and Regulatory Functions of Ligand- and Protein-binding Regulatory RNAs

(A) Riboswitches are composed of an aptamer region (pink) and an expression platform (orange) in the 5’ UTR of an mRNA (blue). Ligand binding can result in transcriptional regulation of mRNA synthesis or translational control of protein synthesis. (Left panel) In the absence of ligand, the expression platform assumes a conformation permissive of transcription—shown here as a stem-loop lacking a U-rich region—allowing synthesis of the entire mRNA. When the ligand binds the aptamer region, a conformational change leads to the disruption of this structure and the formation of an alternative hairpin followed by a string of U residues. This alternative hairpin acts as a transcriptional terminator, inhibiting gene expression. (Middle left panel) In the absence of ligand, the riboswitch initially forms a terminator. Upon ligand binding, this terminator is disrupted, allowing transcription to continue. (Middle right panel) In the absence of ligand, the ribosome binding site (RBS) is accessible, but upon ligand binding, is sequestered into an inhibitory stem-loop, preventing translation. (Right panel) In the absence of ligand, the expression platform forms a repressive secondary structure in which the ribosome binding site is occluded. When the ligand binds to the aptamer region, the ribosome binding site is released and translation can initiate. (B) Protein-binding sRNAs (red) that antagonize regulatory proteins. (Left panel) The CsrA protein (yellow circle) binds to GGA hairpins in mRNAs, altering expression from the transcripts. When CsrB RNA levels increase, the sRNA sequesters CsrA and prevents its regulatory effects. (Middle panel) Under conditions of low 6S abundance, σ⁷⁰-RNA polymerase (blue oval) binds promoter DNA. When 6S levels increase, the sRNA titrates σ⁷⁰- RNA polymerase away from some promoters, reducing transcription of certain housekeeping genes. (Right panel) When GlmY (shorter sRNA) levels are low, YhbJ (green oval) inactivates GlmZ (longer sRNA) by promoting its cleavage. When the GlmY competes with GlmZ for binding to YhbJ, GlmZ is stabilized.

Figure 2. Gene Arrangement and Regulatory Functions of Base Pairing Regulatory RNAs

(A) Two possible configurations of cis-encoded antisense sRNAs (red) and their target RNAs (blue) which share extensive complementarity. (Left panel) An sRNA encoded opposite to the 5’ UTR of its target mRNA. Base pairing inhibits ribosome binding and often leads to target mRNA degradation. (Right panels) An sRNA encoded opposite to the sequence separating two genes in an operon. Base pairing of the sRNA can target RNases to the region and cause mRNA cleavage, with various regulatory effects, or the sRNA can cause transcriptional termination, leading to reduced levels of downstream genes. (B) Genes encoding trans-encoded antisense sRNAs (red) are located separate from the genes encoding their target RNAs (blue) and only have limited complementarity. Trans-encoded sRNA can act negatively by base pairing with the 5’ UTR and blocking ribosome binding (left panel) and/or targeting the sRNA-mRNA duplex for degradation by RNases (middle panel). Trans-encoded sRNA can act positively by preventing the formation of an inhibitory structure, which sequesters the ribosome binding site (RBS) (right panel).

Figure 3. Gene Arrangement and Regulatory Functions of CRISPR RNAs

CRISPR arrays are comprised of DNA repeats (black triangles) separated by unique spacers (red hatched boxes). CAS genes (blue) which encode proteins that function in CRISPR RNA processing and/or DNA silencing are located nearby. The CRISPR arrays are initially transcribed as a long RNA, which is subsequently processed by the Cascade complex (blue circles and ovals) to individual repeat-spacer units, called crRNAs. These crRNAs target foreign DNA through an unknown mechanism likely involving other CAS proteins and the degradation of the exogenous DNA.

Figure 4. Possible Roles of Duplicated RNA Genes

Two homologous sRNAs (red) can act in different manners to regulate mRNAs (blue and purple) and correspondingly alter protein levels (blue and purple circles). (A) Redundant functions. Homologous sRNAs may target the same mRNAs. (B) Additive functions. Multiple sRNAs may precisely control the levels of regulated proteins by each binding to a subset of the target mRNAs. Since relative levels of sRNAs and mRNAs are critical to the effectiveness of regulation, altering the amount of repeated sRNAs can finetune the stability and/or translation of the target mRNAs. (C) Independent functions. Similar sRNAs may use unique sequences to regulate distinct mRNA targets.