Question

1Bacterial genes are regulated by riboswitches that respond to metabolites. Explain how riboswitches regulate gene expression with two examples.

2 Describe the mechanism of regulation of gene expression by miRNA . List two pathways that are disrupted by increase in expression of certain miRNAS. Describe using a specific example,

Answer 1

Answer:

(1) Genetic regulation by RNA is widespread in bacteria. One common form of riboregulation in bacteria is the use of ribonucleic acid sequences encoded within mRNA that directly affect the expression of genes encoded in the full transcript (called cis-acting elements because they act on the same molecule they're coded in). These regulatory elements are known as riboswitches and are defined as mRNA elements that bind metabolites or metal ions as ligands and regulate mRNA expression by forming alternative structures in response to this ligand binding.

A riboswitch can adopt different secondary structures to effect gene regulation depending on whether ligand is bound. This schematic is an example of a riboswitch that controls transcription. When metabolite is not bound (-M), the expression platform incorporates the switching sequence into an antiterminator stem-loop (AT) and transcription proceeds through the coding region of the mRNA. When metabolite binds (+M), the switching sequence is incorporated into the aptamer domain, and the expression platform folds into a terminator stem-loop (T), causing transcription to abort. aptamer domain (red), switching sequence (purple), and expression platform (blue).

The function of riboswitches is tied to the ability of RNA to form a diversity of structures. The most basic of these is the double-stranded helix, similar to that found in DNA. However, since most RNAs, unlike DNA, do not need to maintain perfect Watson-Crick base pairing, they can form other types of structures. For example, a single strand of RNA can fold back on itself to form a hairpin, which is composed of a helix capped by a loop. These elements of structure are called secondary structure. In larger RNAs, the helices and hairpins pack together into a specific pattern, referred to as the tertiary structure.

Riboswitches are most often located in the 5' untranslated region (5' UTR; a stretch of RNA that precedes the translation start site) of bacterial mRNA. There they regulate the occlusion of signals for transcription attenuation or translation initiation. However, not all riboswitches are at the 5' UTR; scientists have discovered that, in some eukaryotic mRNA, the thiamine pyrophosphate (TPP) riboswitch regulates splicing at the 3' end.

Answer:

(2). Discovered in 1993, micoRNAs (miRNAs) are now recognized as one of the major regulatory gene families in eukaryotes. To date, 24521 microRNAs have been discovered and there are certainly more to come. It was primarily acknowledged that miRNAs result in gene expression repression at both the level of mRNA stability by conducting mRNA degradation and the level of translation (at initiation and after initiation) by inhibiting protein translation or degrading the polypeptides through binding complementarily to 3′UTR of the target mRNAs.

Nevertheless, some studies revealed that miRNAs have the capability of activating gene expression directly or indirectly in respond to different cell types and conditions and in the presence of distinct cofactors. This reversibility in their posttranslational gene regulatory natures enables the bearing cells to rapidly response to different cell conditions and consequently block unnecessary energy wastage or maintain the cell state.

miRNAs are indeed able to reduce gene expression by multiple pathways and modes:-

1st Pathway: Potential mechanisms of miRNA-mediated downregulation. Ribonucleoproteins (miRNPs) target mRNAs through sequence complementation. The association between miRNPs and mRNAs can have one or several consequences. miRNP-mediated downregulation may have direct and indirect mechanisms and occur before translation is triggered or after translation initiation. (a–c) Translation initiation mechanisms. miRNP interferes with early steps of translation before elongation. (a) miRNPs recruit several factors and enzymes for mRNA cleavage and degradation including decapping enzymes, deadenylase, 3′ and 5′ exonucleases, and endonucleases. (b, c) Argonaute protein competes with cap binding proteins (CBPs) and elf4E for binding to cap structure and inhibits translation initiation by interfering with mRNA circularization and formation of closed-loop achieved through cap structure interaction with CBPs and elf4E/G required for translation initiation. (d–h) Postinitiation mechanisms. miRNPs repress translation elongation and termination or involve in protein degradation and sequestration. (d, e) miRNP interferes with ribosome subunit by inhibiting its joining or promoting its dissociation. (f, g) miRNP obstructs translation elongation by competing with elongation factors or cotranslationally recruiting protein decay factors such as exosomes. (h) Target mRNA is sequestered from translation machinery and stored or sometimes is degraded by enzymes. However, alternatively, translationally inhibited mRNA along with associated proteins could be sequestered at the same bodies.

2nd Pathway: In contrast to general assumption that miRNA-mediated downregulation is a one-way process and leads to decreased mRNA stability and/or translational inhibition, Vasudevan and Steitz reported for the first time that the miRNA-mediated downregulation is reversible. Likewise, there is evidence suggesting that some miRNAs could upregulate gene expression in specific cell types and conditions with distinct transcripts and proteins.

In miRNA-mediated upregulation, miRNPs act in trans in promoting their target mRNAs’ expression similar to miRNA-mediated downregulation. The mRNA expression could be activated by the direct action of miRNPs and/or could be indirectly relieved from miRNA-mediated repression by abrogating the action of repressive miRNPs.

miRNA-mediated upregulation according to cell cycle phase. miR-334 and miR-27a bind to the 3′UTR of KLF4 and Myt1 mRNA in proliferating and cancerous cells and subsequently downregulate mRNA expression by both translational inhibition and mRNA degradation. miRNP-mediated repression requires factors including AGO, GW182, deadenylases, and nucleases. However, in G0 cells and immature oocytes, GW182 is downregulated and FXR1 is associated with AGO2 bearing miRNP and leads to gene expression upregulation. miR-206 is associated with KLF4 mRNA in G0 cells and xmiR-16 is associated with Myt1 mRNA in immature Xenopus oocyte. miR-206 and xmiR-16 activate KLF4 and Myt1 expression, respectively, by binding to their 3′UTRs and recruiting special factors such as FXR1, since AGO2 is associated with gene expression activation in these cells and lacks slicer activity.