MICRB 265 LECTURE 4-4
Post-transcriptional Regulation
Lecture Overview
An overview of the regulation that occurs after transcription initiation in bacteria.
Key mechanisms involved include:
RNA-based regulatory mechanisms: sRNAs and riboswitches.
Post-translational regulation.
Reference Textbook: Sections 7.12, 7.13, 7.15, 7.16.
Regulation at the Level of RNA
Overview: A significant amount of gene regulation is conducted after transcription begins but before protein translation occurs.
Key Regulatory Processes
Transcriptional Attenuation: This involves the termination of mRNA synthesis before the associated genes are fully transcribed, preventing unnecessary expression.
mRNA Stability: RNA stability controls the duration of mRNA before degradation. The longer the mRNA persists, the higher the translation rates and resultant protein output are.
Translation Efficiency: Regulation can occur based on whether the ribosome binding site (RBS) is accessible for ribosome attachment. The structure of the RBS plays a crucial role in this regard.
Execution of Regulation: Much of this regulation is facilitated by RNA regulatory elements.
mRNA Lifetime Effects Protein Levels
RNA Degradation Mechanisms
According to Jester et al., Int. Jour. Micro., 2011:
Multiple ribonucleases (RNases) exist in all cells and are responsible for degrading mRNAs. They detect differing sequence or structural characteristics of mRNAs and degrade them accordingly.
The targeting of mRNAs by RNases is complex, and not all mRNAs are formatively targeted consistently.
Regulatory proteins and RNAs can influence mRNA half-lives.
Bacterial mRNA Half-lives: Bacterial transcripts typically have short half-lives, ranging from mere seconds to around an hour, with many existing predominantly in the few-minutes range.
RNA Structures that “Shut Off” Gene Expression
Intrinsic Terminators: During transcription, certain RNA structures, particularly intrinsic transcriptional terminators, may form within the 5' UTR before transcription completes, leading to transcriptional attenuation.
RBS Accessibility: For initiation of translation, the ribosome binding site (RBS) must be unbound and accessible; pairing in mRNA sequences can block access.
Stem-loop Structures: Formation of stem-loop structures in mRNA can inhibit gene expression if:
They disrupt the RBS, or
They facilitate upstream transcriptional terminators.
sRNAs (Small Regulatory RNAs)
Definition: All cells produce regulatory RNAs, which are noncoding RNAs designed to modulate gene expression.
Characteristics of Bacterial sRNAs:
Typically range between 50 and 300 nucleotides.
Generally operate by base-pairing with target mRNAs, affecting RBS availability and/or the targeting by RNases, which influences protein synthesis levels.
A single sRNA can regulate multiple mRNA targets.
sRNAs and Hfq Protein
Sequence Complementarity: Many sRNAs have limited sequence complementarity, often only a 5-11 nucleotide stretch in base pairing with target mRNAs.
Role of Hfq: Hfq acts as an RNA chaperone essential for facilitating the interactions between sRNAs and mRNAs.
It binds both RNAs to fine-tune their interactions, and in certain cases, it also recruits RNases to initiate degradation of target mRNAs.
Riboswitches
Overview and Function
Definition: Riboswitches are segments of mRNA located in the 5' UTR, having the capacity to regulate gene expression via ligand binding.
Structure: They contain aptamers that specifically bind to small molecules, enabling riboswitches to monitor cellular conditions.
Expression Regulation: The interaction with ligands alters RNA structure within the 5' UTR, which subsequently either induces or represses the expression of downstream genes. This process can flip expression states from “on” to “off” and vice versa.
Distribution: Riboswitches have been identified across all three domains of life but are predominantly characterized in bacteria.
Mechanisms of Riboswitch Action
Tracking Changes: Ligand binding modifies base-pairing behaviors in the 5' UTR, influencing the formation of:
Stem-loop structures that sequester the RBS (blocking translation), or
Transcriptional terminators that inhibit transcription of effector genes.
Evolutionary Significance of Riboswitches
Hypotheses: Riboswitches are considered “relics from the RNA world.” They are believed to have been significant in the environment where RNA was central to life.
Metabolite Binding: They can bind a variety of central metabolites indicative of environmental sensing capabilities, showing conserved aptamer forms across divergent lineages, suggesting ancient ancestry.
Examples of Riboswitch Ligands
Riboswitches are known to interact with various metabolites crucial for the early RNA world.
Post-translational Regulation
Overview of Mechanisms
Regulation of protein levels or activities can occur via:
Feedback Inhibition: Enzymes can be inhibited by the end products of their respective biosynthetic pathways.
Protein-Protein Interactions: The activity of proteins can be modulated through interactions with other proteins.
Post-translational Modifications: The addition of chemical moieties, such as phosphate groups, to specific amino acid residues alters protein activity, effectively turning it “on” or “off.”
Protein Degradation and Turnover
Key Functions of Proteases
Proteins, like RNAs, can also undergo degradation and recycling through proteases.
Importance of Proteases:
They are essential for clearing misfolded proteins and regulating cellular protein proportions by specifically targeting proteins for degradation.
Cleavage by proteases can activate certain proteins by separating peptide linkages, crucial for proper functionality.
Reference for proteolytic activity: Activity of Lon, a prevalent bacterial protease as reported by Puri & Karzai, Molecular Cell, 2017.
Summary of Regulatory Mechanisms
An illustrative overview of regulatory mechanisms is depicted in Textbook Figure 7.4. Key Insight: Regulation of gene expression is only meaningful in the context of environmental sensing and response characteristics in cellular systems.
Post-transcriptional Regulation Summary
Regulation occurs at various levels, starting from DNA through protein levels and activity:
Beginning of Transcription: Regulation can occur at the initiation of transcription. (Though the provided notes focus on post-transcriptional regulation, the overall context implies this initial stage).
Regulation at the Level of RNA (Post-transcriptional): This is a significant amount of gene regulation conducted after transcription begins but before protein translation occurs. Key processes include transcriptional attenuation, mRNA stability, and translation efficiency.
Post-translational Regulation: Regulation of protein levels or activities after translation, involving feedback inhibition, protein-protein interactions, and post-translational modifications.
Regulation at the Level of RNA and Major RNA Structures
Gene expression is regulated at the level of RNA through several major ways:
Transcriptional Attenuation: This involves the termination of mRNA synthesis prematurely, preventing full transcription of associated genes. Major RNA structures involved are:
Intrinsic Terminators: Specific RNA structures, often forming within the 5' UTR during transcription, which can lead to premature transcription termination.
mRNA Stability: Control over the duration mRNA persists before degradation. Longer mRNA half-lives lead to higher translation rates and protein output.
Translation Efficiency: Regulation based on the accessibility of the ribosome binding site (RBS) for ribosome attachment. Major RNA structures involved are:
Stem-loop Structures: Formation of these structures in mRNA can inhibit gene expression if they disrupt the RBS or facilitate upstream transcriptional terminators, thereby blocking ribosome access or preventing transcription altogether.
sRNAs (Small Regulatory RNAs): Noncoding RNAs (50-300 nucleotides) that typically operate by base-pairing with target mRNAs, affecting RBS availability and/or targeting by RNases, thus influencing protein synthesis.
Riboswitches: Segments of mRNA in the 5' UTR that regulate gene expression by binding small molecules (ligands), altering RNA structure to induce or repress downstream gene expression.
RNA Lifetime and Controlling Factors
RNA lifetime, or mRNA stability, refers to the duration an mRNA molecule persists in the cell before it is degraded. The longer the mRNA persists, the higher the rates of translation and subsequent protein production.
Factors controlling RNA lifetime include:
Ribonucleases (RNases): Multiple types of RNases exist in all cells, responsible for degrading mRNAs. They detect differing sequence or structural characteristics of mRNAs and degrade them accordingly.
mRNA Characteristics: The specific sequence or structural features of mRNAs can influence how they are targeted by RNases.
Regulatory Proteins and RNAs: These can influence mRNA half-lives, either extending or shortening them.
Bacterial transcripts typically have short half-lives, ranging from mere seconds to about an hour, with many lasting only a few minutes.
Small Regulatory RNAs (sRNAs) and their Mechanisms
sRNAs are noncoding regulatory RNAs produced by all cells, designed to modulate gene expression. In bacteria, they typically range between 50 and 300 nucleotides.
sRNAs generally control gene expression through the following mechanisms:
Base-pairing with Target mRNAs: They operate by base-pairing with specific stretches (often 5-11 nucleotides) of target mRNAs.
Affecting RBS Availability: This base-pairing can either block or unblock the ribosome binding site (RBS), thereby inhibiting or promoting translation initiation.
Influencing RNase Targeting: sRNAs can also affect whether target mRNAs are destined for degradation by RNases, thus altering mRNA stability and protein synthesis levels.
A single sRNA can regulate multiple mRNA targets, making them versatile regulators.
Function of Hfq in sRNA-mediated Regulation
Hfq is an RNA chaperone essential for facilitating the interactions between sRNAs and their target mRNAs. Many sRNAs have limited sequence complementarity with their target mRNAs (often only a 5-11 nucleotide stretch), making their interaction somewhat weak or transient. Hfq plays a crucial role by:
Binding Both RNAs: It binds both the sRNA and the target mRNA, helping to 'fine-tune' and stabilize their interactions.
Recruiting RNases: In certain cases, Hfq also recruits RNases to the sRNA-mRNA complex, leading to the degradation of the target mRNA and further regulation of gene expression.
Definition of Riboswitch and Differences from sRNAs
A riboswitch is a segment of mRNA located in the 5' UnTranslated Region (5' UTR) that has the capacity to regulate gene expression via ligand binding. Riboswitches contain aptamers that specifically bind to small molecules, allowing them to monitor cellular conditions and adjust gene expression accordingly.
Key differences between riboswitches and sRNAs include:
Location and Mechanism: Riboswitches are cis-acting elements, meaning they are integral parts of the mRNA molecule they regulate (located in its 5' UTR) and directly respond to ligand binding by altering their own structure. sRNAs, on the other hand, are trans-acting regulators; they are separate RNA molecules that typically diffuse and base-pair with target mRNAs elsewhere in the cell.
Regulatory Input: Riboswitches directly bind small molecule ligands (metabolites) to sense cellular conditions. sRNAs do not directly bind ligands; their activity is often regulated by other factors or cellular signals that control their expression or stability.
Mode of Action: Riboswitches operate through conformational changes induced by ligand binding that affect transcription termination or translation initiation of the same mRNA. sRNAs operate by base-pairing with other mRNA molecules, affecting their stability or translation.
Mechanisms of Riboswitch Regulation
Riboswitches regulate gene expression through mechanisms that involve ligand binding modifying base-pairing behaviors in the 5' UTR, which is common to all riboswitch-mediated regulation. This structural alteration subsequently either induces or represses the expression of downstream genes, essentially flipping expression states from “on” to “off” or vice versa.
What differs among individual riboswitches depends on how this structural change impacts gene expression:
Sequestering the RBS: Ligand binding can induce the formation of stem-loop structures that sequester (hide) the ribosome binding site (RBS), thereby blocking translation initiation.
Forming Transcriptional Terminators: Ligand binding can promote the formation of transcriptional terminators within the 5' UTR, which leads to premature termination of transcription and inhibits the transcription of effector genes.
Evolutionary Significance of Riboswitches
Riboswitches are of significant evolutionary interest and are considered “relics from the RNA world.” This hypothesis suggests that they played a crucial role in an environment where RNA was central to life, before the dominance of DNA and proteins.
Their potential role in the early stages of (pre-)life includes:
Metabolite Binding: Their ability to bind a variety of central metabolites indicates ancient environmental sensing capabilities. This suggests that early life forms, potentially relying heavily on RNA for both genetic information and catalysis, could have used riboswitches to directly sense and respond to the availability of essential nutrients or regulatory molecules.
Conserved Aptamer Forms: The presence of conserved aptamer forms across divergent lineages of life suggests a very ancient ancestry, supporting the idea that these molecular switches were fundamental to early life's regulatory machinery.
Post-translational Regulation at the Protein Level
Regulation can occur at the protein level in several different ways, affecting protein levels or activities:
Feedback Inhibition: Enzymes are inhibited by the end products of their respective biosynthetic pathways. This allows cells to halt production when sufficient product is available.
Protein-Protein Interactions: The activity of proteins can be modulated through direct interactions with other proteins.
Protein Degradation and Turnover
Key Functions of Proteases
Proteins, like RNAs, can also undergo degradation and recycling through proteases.
Importance of Proteases:
They are essential for clearing misfolded proteins (housekeeping function) and regulating cellular protein proportions by specifically targeting proteins for degradation (regulatory function).
Cleavage by proteases can activate certain proteins by separating peptide linkages, crucial for proper functionality (regulatory function).