RNA-Based Regulation

RNA Regulation and Antisense RNA

Antisense RNAs are single-stranded RNA molecules that can bind and inhibit specific mRNA translation to protein.

Learning Objectives

Explain RNA regulation via antisense RNA

Key Takeaways

Key Points

  • Antisense RNAs are specific to mRNAs based on the principle of complementary base pairs.
  • Antisense RNAs bind to mRNAs and inhibit the ability of these mRNAs to be translated into functioning protein.
  • Naturally occurring antisense RNAs have been identified in E. coli, which carry an R1 plasmid and control the hok/sok system that involves the production of toxic and antitoxic products.

Key Terms

  • morphogenesis: The differentiation of tissues and growth in organisms.

Gene regulation, the ability to control whether a gene is expressed or not, is critical in controlling cellular and metabolic processes and contributes to diversity and variation in organisms. Furthermore, it is the key determinant in cellular differentiation and morphogenesis. There are specific types of RNA molecules that can be utilized to control gene regulation, including messenger RNAs (mRNAs), small RNAs such as microRNAs and lastly, antisense RNAs. The following is a brief overview of antisense RNAs and their role in RNA regulation. Antisense RNAs have been recently investigated as a new class of antiviral drugs.

Antisense RNAs are single-stranded RNA molecules that exhibit a complementary relationship to specific mRNAs. Antisense RNAs are utilized for gene regulation and specifically target mRNA molecules that are used for protein synthesis. The antisense RNA can physically pair and bind to the complementary mRNA, thus inhibiting the ability of the mRNA to be processed in the translation machinery. Pairing antisense RNA is a technique that can be utilized within the laboratory for gene regulation — however, it is not without limitations. Naturally occurring antisense RNAs have been isolated in a various microbes, including the E. coli RI plasmid, which uses a hok/sok system. A hok/sok system is a mechanism employed by E. coli that is used as a postsegregational killing mechanism. The hok gene is a toxic gene and the sok gene is an antitoxin. Hence, E. coli utilizing this system can regulate the expression of hok (toxin) and inhibits its translation by producing sok RNA (antitoxin). The outcome is the repression of hok mRNA translation.

image

Hok/sok System: An example of a system found in nature that utilizes an antisense RNA to control gene regulation. The hok gene is a toxic gene that can be translationally repressed by the production of an antisense mRNA, sok (anti toxic).

Attenuation

Attenuation is a mechanism utilized by bacteria to regulate unnecessary gene expression.

Learning Objectives

Compare transcriptional and translational attenuation

Key Takeaways

Key Points

  • Attenuators are characterized by the presence of stop signals within the DNA sequence that can result in either transcriptional- attenuation or translational-attenuation.
  • Transcriptional-attenuation is characterized by the presence of an attenuator within the DNA sequence that results in formation of mRNA-stem loops that prevent further transcription from occurring. The non-functional RNA produced prevents proper transcription.
  • Translational-attenuation is characterized by the misfolding of the Shine-Dalgarno sequence. The Shine-Dalgarno sequence, responsible for ribosomal binding to allow proper translation, is inaccessible because it is folded into a hairpin-loop structure, thus, translation cannot occur.

Key Terms

  • operons: A unit of genetic material that functions in a coordinated manner and is transcribed as one unit.

Attenuation is a regulatory mechanism used in bacterial operons to ensure proper transcription and translation. In bacteria, transcription and translation are capable of proceeding simultaneously. The need to prevent unregulated and unnecessary gene expression can be prevented by attenuation, which is characterized as a regulatory mechanism.

image

Attenuation of the Tryptophan Operon: An example of attenuation is the tryptophan operon. This schematic represents transcriptional-attenuation as the formation of mRNA stem-loops prevents the continuance of transcription based on the levels of tryptophan in the metabolic environment.

The process of attenuation involves the presence of a stop signal that indicates premature termination. The stop signal, referred to as the attenuator, prevents the proper function of the ribosomal complex, stopping the process. The attenuator is transcribed from the appropriate DNA sequence and its effects are dependent on the metabolic environment. In times of need, the attenuator within the mRNA sequence will be bypassed by the ribosome and proper translation will occur. However, if there is not a need for a mRNA molecule to be translated but the process was simultaneously initiated, the attenuator will prevent further transcription and cause a premature termination. Hence, attenuators can function in either transcription-attenuation or translation-attenuation.

Transcription-attenuation is characterized by the presence of 5′-cis acting regulatory regions that fold into alternative RNA structures which can terminate transcription. These RNA structures dictate whether transcription will proceed successfully or be terminated early, specifically, by causing transcription-attenuation. The result is a misfolded RNA structure where the Rho-independent terminator disrupts transcription and produced a non-functional RNA product. This characterizes the mechanisms of transcription-attenuation. The other RNA structure produced will be an anti-terminator that allows transcription to proceed.

Translation-attenuation is characterized by the sequestration of the Shine-Dalgarno sequence. The Shine-Dalgarno sequence is a bacterial specific sequence that indicates the site for ribosomal binding to allow for proper translation to occur. However, in translation-attenuation, the attenuation mechanism results in the Shine-Dalgarno sequence forming as a hairpin-loop structure. The formation of this hairpin-loop structure results in the inability of the ribosomal complexes to form and proceed with proper translation. Hence, this specific process is referred to as translation-attenuation.

Riboswitches

Riboswitches are naturally occurring RNA molecules that can regulate gene expression.

Learning Objectives

Describe riboswitches

Key Takeaways

Key Points

  • The mechanisms by which riboswitches regulate RNA expression, can be divided into two major processes, including aptamer and expression platform.
  • The aptamer is characterized by the direct binding of the small molecule to its target.
  • The expression platform is characterized by the conformational change, which occurs in the target upon binding of an aptamer, resulting in either inhibition or activation of gene expression.

Key Terms

  • aptamer: Any nucleic acid or protein that is used to bind to a specific target molecule.
image

Example of a Riboswitch: A 3D image of the riboswitch responsible for binding to thiamine pyrophosphate (TPP)

Riboswitches are specific components of an mRNA molecule that regulates gene expression. The riboswitch is a part of an mRNA molecule that can bind and target small target molecules. An mRNA molecule may contain a riboswitch that directly regulates its own expression. The riboswitch displays the ability to regulate RNA by responding to concentrations of its target molecule. The riboswitches are naturally occurring RNA molecules that allow for RNA regulation. Hence, the existence of RNA molecules provide evidence to the RNA world hypothesis that RNA molecules were the original molecules, and that proteins developed later in evolution.

Riboswitches are found in bacteria, plants, and certain types of fungi. The various mechanisms by which riboswitches function can be divided into two major parts including an aptamer and an expression platform. The aptamer is characterized by the ability of the riboswitch to directly bind to its target molecule. The binding of the aptamer to the target molecule results in a conformational change of the expression platform, thus affecting gene expression. The expression platforms, which control gene expression, can either be turned off or activated depending on the specific function of the small molecule. Various mechanisms by which riboswitches function include, but are not limited to the following:

  • The ability to function as a ribozyme and cleave itself if a sufficient concentration of its metabolite is present
  • The ability to fold the mRNA in such a way the ribosomal binding site is inaccessible and prevents translation from occurring
  • The ability to affect the splicing of the pre-mRNA molecule

The riboswitch, dependent on its specific function, can either inhibit or activate gene expression.

Regulation of Sigma Factor Activity

The sigma factor is responsible for proper transcriptional initiation.

Learning Objectives

Analyze the regulation of sigma factor activation

Key Takeaways

Key Points

  • Sigma factor proteins promote binding of RNA polymerase to promoter sites within DNA sequences to allow for initiation of transcription.
  • Sigma factors are specific for the gene and are affected by the cellular environment.
  • Sigma factors can regulate at both a transcription and translational level.
  • Anti-sigma factors are responsible for inhibiting sigma factor function thus, inhibiting transcription.

Key Terms

  • growth phase transitions: The various phases required for bacterial growth include: lag, exponential, and stationary phases.

Sigma factors are proteins that function in transcription initiation. Specifically, in bacteria, sigma factors are necessary for recognition of RNA polymerase to the gene promoter site. The sigma factor allows the RNA polymerase to properly bind to the promoter site and initiate transcription which will result in the production of an mRNA molecule. The type of sigma factor that is used in this process varies and depends on the gene and on the cellular environment. The sigma factors identified to date are characterized based on molecular weight and have shown diversity between bacterial species as well. Once the role of the sigma factor is completed, the protein leaves the complex and RNA polymerase will continue with transcription.

image

Sigma factor SigR: Structure of sigma factor.

The regulation of sigma factor activity is critical and necessary to ensure proper initiation of transcription. The activity of sigma factors within a cell is controlled in numerous ways. Sigma factor synthesis is controlled at the levels of both transcription and translation. Often times, sigma factor expression or activity is dependent on specific growth phase transitions of the organism. If transcription of genes involved in growth is necessary, the sigma factors will be translated to allow for transcription initiation to occur. However, if transcription of genes is not required, sigma factors will not be active.

In specific instances when transcriptional activity needs to be inhibited, there are anti-sigma factors which perform this function. The anti-sigma factors will bind to the RNA polymerase and prevent its binding to sigma factors present at the promoter site. The anti-sigma factors are responsible for regulating inhibition of transcriptional activity in organisms that require sigma factor for proper transcription initiation.

Regulation of Sigma Factor Translation

Sigma factors are proteins that regulate gene expression that are controlled at various levels, including at the translational level.

Learning Objectives

Explain the regulation of sigma factor translation

Key Takeaways

Key Points

  • Sigma factor expression is often associated with environmental changes that cause changes in gene expression.
  • The translational control of sigma factors is critical in its role in transcription regulation.
  • Sigma factor translation is controlled by small noncoding RNAs that can either activate or inhibit translation.

Key Terms

  • oxidative stress: Damage caused to cells or tissue by reactive oxygen species.
  • sigma factor: A sigma factor (σ factor) is a protein needed only for initiation of RNA synthesis.
  • RpoS protein: RpoS is a central regulator of the general stress response and operates in both a retroactive and a proactive manner: not only does it allow the cell to survive environmental challenges, but it also prepares the cell for subsequent stresses (cross-protection).

Sigma factors are groups of proteins that regulate transcription and therefore function in house-keeping, metabolic, and regulation of growth processes in bacteria. Sigma factor expression is often associated with environmental changes that cause changes in gene expression. The regulation of expression of sigma factors occurs at transcriptional, translational, and post-translational levels as dictated by the cellular environment and the presence or absence of numerous cofactors.

image

Sigma Factor Regulation of Transcription: An overview of how sigma factors regulate the transcriptional process.

Sigma factors include numerous types of factors. The most commonly studied sigma factors are often referred to as a RpoS proteins as the rpoS genes encode for sigma proteins of various sizes. In E. coli, the RpoS is the regulator of growth phase genes, specifically in the stationary phase. The RpoS is critical in the general stress responses and can either function in promoting survival during environmental stresses, but can also prepare the cell for stresses. Specifically, the translational control of the sigma factor is a major level of control.

The translational control of sigma factors involves the presence and function of small noncoding RNAs. Using RpoS proteins as the focus, the RpoS expression and transcription is regulated at the translational level. Small noncoding RNAs are able to sense environmental changes and stresses resulting in increased expression of RpoS protein. The small noncoding RNAs are able to specifically increase the amount of rpoS mRNA that undergoes translation.

The resultant increase of RpoS protein is based on the cellular environment and its needs. There are numerous classes of small noncoding RNAs that function in RpoS regulation, including DsrA, RprA and OxyS. These small noncoding RNAs are capable of sensing changes in temperature (DsrA), cell surface stress (RprA) and oxidative stress (OxyS). These RNAs can induce activation of rpoS translation. However, there are small noncoding RNAs, such as LeuO, that are capable of inhibiting rpoS translation as well via repression mechanisms. The regulation of rpoS translation is complex and involves cross-signaling and networking of numerous proteins and the regulatory small noncoding RNAs.

Proteolytic Degradation

Proteolytic degradation, or proteolysis, is a key factor that controls protein concentration and function.

Learning Objectives

Describe protein degradation

Key Takeaways

Key Points

  • The major mechanism of proteolytic degradation utilized by the cell, is via the proteasomal pathway. Proteins that are degraded via the proteasomal complex are tagged via the addition of a ubiquitin signal.
  • An additional mechanism utilized for proteolytic degradation is via the lysosomal pathway. The lysosome contains proteases which target proteins for degradation.
  • Proteolysis is necessary to control protein concentration and prevent abnormal accumulation.
  • Upon protein degradation, the amino acids are typically reused and recycled for the synthesis of new proteins.

Key Terms

  • ubiquitin: A small regulatory protein sequence that directs proteins to specific compartments within the cell. Specifically, a ubiquitin tag directs the protein to a proteasome, which destroys and recycles the components.
  • proteases: A class of enzymes that can cleave proteins.
image

The Process of Protein Degradation in a Proteosome: Schematic of the proteolytic degradation pathway that utilizes proteasomal complexes. The protein is tagged with several ubiquitin signals that target the proteasome. Once the protein arrives at the proteasome, the protein is degraded into its amino acids which are then reused for synthesis of new proteins.

Proteolytic degradation is necessary in the regulation of cellular processes and function. The breakdown of proteins into smaller polypeptides, or its respective amino acids, are necessary for metabolic and cellular homeostasis. Polypeptides are commonly broken down via hydrolysis of the peptide bonds by utilizing a class of enzymes called proteases. However, proteolytic degradation can also occur utilizing various mechanisms, including intramolecular digestion and non-enzymatic methods. The mechanisms of proteolytic degradation are necessary for obtaining amino acids via degradation of digested proteins, preventing accumulation or abnormal concentrations of proteins, and by regulating cellular processes by removing proteins no longer needed.

Proteasomes are protein complexes that function in the degradation of unneeded or damaged proteins via proteolysis. The proteasomes are a major component of a complex and highly regulated mechanism. The proteasome is able to degrade proteins based on the presence of a ubiquitinprotein. This ubiquitin sequence is a modification to proteins that are targeted for degradation. The recognition of this ubiquitin signal by the proteasome results in degradation of the protein into its amino acids, which are then recycled and reused for the synthesis of new proteins. The proteasomal degradation pathway is the major pathway utilized to ensure proteolytic degradation. It is necessary for homeostasis functioning in controlling cell cycle and gene expression, for example.

In addition to proteasomal complexes, the organelle, the lysosomes are also used to ensure protein degradation. The intracellular process that utilizes lysosomes involves autophagy. The lysosomal pathway, in comparison to the proteasomal pathway, is typically non-selective. The lysosome contains proteases that are able to target and degrade proteins.

Small Regulatory RNAs

Small regulatory RNAs are non-coding RNA molecules that play a role in cellular processes such as activation or inhibition processes.

Learning Objectives

Recall the types of small regulatory RNAs

Key Takeaways

Key Points

  • Small regulatory RNAs are highly structured and contain several stem-loops that contribute to function allowing for binding to targets.
  • Classes of small regulatory RNAs include antisense RNAs, house-keeping genes, outer membrane protein regulation, and regulation of RPoS.
  • Small regulatory RNAs can function by activating or inhibiting translation processes.

Key Terms

  • RNA polymerase: An enzyme responsible for the synthesis of RNA during transcription.

Small regulatory RNAs encompass a specific class of RNAs that affect gene regulation. These RNAs are typically small non-coding RNA molecules that are highly structured and are comprised of numerous stem-loops. These small regulatory RNAs play a critical role in gene regulation via numerous mechanisms. The mechanisms by which small regulatory RNAs function include binding to protein targets, protein modification, binding to mRNA targets, and regulating gene expression. There are numerous classes of small regulatory RNAs that play a key role in regulation.

image

Overview of Antisense DNA: This image displays a mechanism of antisense DNA. However, it is important to note that an antisense RNA functions in the same manner. The antisense RNA can bind to the mRNA and inhibit translation. In some cases, small regulatory RNAs, not included in the antisense category, can activate translation as well.

Antisense RNAs

Antisense RNAs are used to bind to complementary mRNAs and inhibit protein translation. Antisense RNAs are single stranded RNAs that can be utilized as a laboratory technique to inhibit protein translation. Antisense RNAs have also been found to be naturally occurring in bacteria such as E. coli with the R1 plasmid. The antisense RNAs are categorized as small regulatory RNAs due to their small size. They can be divided into either cis- or trans-antisense RNAs. Cis-antisense RNAs are encoded by an overlap between the antisense RNA itself and the target gene. In trans-antisense RNAs, the antisense RNA gene is separate from the target gene and there is no overlap.

House-keeping RNAs

Small regulatory RNAs encompass many RNAs involved in house-keeping processes as well. House-keeping genes are specific genes that function in maintaining basic cellular processes and a state of homeostasis. House-keeping RNAs identified to date include rRNA and tRNAs. rRNAs that are considered to be house-keeping genes can bind to RNA polymerases and regulate transcription or function in larger complexes that are required for protein secretion or synthesis processes.

Regulation of RPoS

RPoS genes specifically encode for sigma factors which function as regulators of transcription and stress responses. Small RNAs have been shown to regulate RPoS translation and those identified thus far include: DsrA, RprA, and OxyS. These RNAs can either activate or inhibit RPoS translation.

Outer Membrane Protein Regulation

Small regulatory RNAs have also been identified in regulation of outer membrane proteins that function in critical cellular processes such as controlling entry and exit to the cell. The outer membrane functions as a barrier for the cell and its ability to allow for transport is strictly controlled. For example, the small regulatory RNAs, MicC, and MicF, are able to regulate expression of outer membrane proteins (OmpC and OmpF) which function in controlling transport of metabolites and toxins.