In this tutorial, you will learn about introns and exons, including their definitions, roles in gene expression, and differences. You will explore the process of RNA splicing, focusing on the mechanisms of Group I, II, and III introns. Additionally, you’ll discover exon shuffling and its significance in genetic diversity, and gain insights into ribozymes, including their catalytic properties and types such as hammerhead and hairpin ribozymes.
Contents:
- Introduction to Introns and Exons
- RNA Splicing: Removal of Introns
- Group II and III Introns: Spliceosome-Mediated Splicing
- Exon Shuffling
- What Are Ribozymes?
- Hammerhead Ribozymes
- Hairpin Ribozymes
Introduction to Introns and Exons
Introns and exons are essential components of genes in eukaryotic cells that play a crucial role in gene expression and protein synthesis.
- Exons are the coding sequences of DNA that are expressed in the final mRNA product. These segments of DNA are transcribed into mRNA and later translated into proteins.
- Introns, on the other hand, are non-coding regions of DNA found between exons. They do not contribute directly to protein synthesis and are removed during RNA processing.
In a typical human gene, there are about 7.9 introns, which is fewer compared to the number of exons. However, despite not coding for proteins, introns are vital for regulating gene expression and the splicing process.
RNA Splicing: Removal of Introns
RNA splicing is the process through which introns are removed from pre-mRNA, and exons are joined together to form mature mRNA. This is a crucial step in the process of gene expression because only mature mRNA is translated into functional proteins. There are different types of introns, each with its own unique splicing mechanism.
Group I Introns: Self-Splicing Mechanism
Group I introns are found in certain genes, such as those in the mitochondria, chloroplasts, and nuclei of some organisms. These introns exhibit a self-splicing mechanism that doesn’t require protein enzymes.
Mechanism:
- A guanosine cofactor initiates a nucleophilic attack on the 5′ splice site, adding guanosine to the 5′ end of the intron and releasing the 5′ exon.
- This is followed by a conformational rearrangement where the 5′ exon attacks the 3′ exon, resulting in ligation of the exons and the release of the intron as a linear molecule.
- The entire reaction is energy-independent, meaning no ATP is required for splicing.
Group II and III Introns: Spliceosome-Mediated Splicing
Group II and III introns, found in nuclear mRNA transcripts, require the spliceosome, a complex of snRNPs (small nuclear ribonucleoproteins), for splicing. The splicing mechanism involves lariat formation:
Mechanism:
- The 5′ splice site (GU) and the 3′ splice site (AG) are recognized by U1 and U2 snRNPs, respectively.
- U2 snRNA pairs with the intron at a specific A residue, which acts as the nucleophile.
- A lariat structure forms through a 2′,5′-phosphodiester bond between the branch point A and the 5′ splice site.
- The spliceosome undergoes conformational changes to activate the complex, excise the intron, and join the exons.
Exon Shuffling
Exon shuffling is a genetic mechanism where exons from different genes are rearranged to create new combinations. This process increases genetic diversity and contributes to the evolution of new proteins with novel functions.
- An existing eukaryotic gene generates combinatorial library. Combinatorial library is a place where mixtures of biological, chemical, or synthetic compounds are kept and stored. The reason of using combinatorial library is to generate more diversified molecular compounds for further discoveries.
- PCR reaction then generates separate exons of genes, and these segments are mixed and reassembled by PCR overlapping. This reassembling is done in a random manner, and this creates random splicing library.
- Products are then screened for obtaining long sequences for encoding original exons and this enhances chances of obtaining a fully functional protein.
What Are Ribozymes?
Ribozymes are RNA molecules that have catalytic properties, meaning they can catalyze chemical reactions, similar to enzymes. They play various roles in RNA processing, including the splicing of introns.
Key Characteristics of Ribozymes:
- Catalytic Function: Ribozymes can facilitate biochemical reactions, such as the cleavage and ligation of RNA.
- Self-Splicing: Some ribozymes are capable of self-splicing, meaning they can remove their own introns without the need for additional proteins.
- Biological Significance: Ribozymes are involved in various cellular processes, including RNA processing, gene regulation, and the replication of some viruses.
Hammerhead Ribozymes
Hammerhead ribozymes catalyze the cleavage and ligation of RNA molecules. They are unique because they don’t require proteins for catalysis.
Cleavage reaction:
- This cleavage reaction is a phosphodiester isomerization reaction, initiated by abstraction of cleavage site ribose 2-hydroxyl proton from oxygen which becomes attacking nucleophile.
- A general base abstracts a proton from 2’-O and a general acid supplies proton to 5’O leaving group as negative charge accumulates. The reaction product is 2’,3’-cyclic phosphate.
- As a result of cleavage of reaction mechanism, 5’product possesses a 2’,3’-cyclic phosphate terminus and 3’product possesses a 5’OH terminus.
- Cleavage takes place in absence of protein enzymes, but hammerhead ribozymes is not a catalyst itself, as it is consumed by reaction (self-cleavage) and therefore cannot catalyze multiple turnovers.
Hairpin Ribozymes
Hairpin ribozymes are found in the replication cycles of certain viral RNAs, such as the Tobacco ringspot virus (TRVS) satellite RNA. Unlike many other ribozymes, hairpin ribozymes do not require metal ions for catalysis.
Significance of hairpin ribozymes in catalysis process:
- Hairpin ribozyme-substrate complex folds into a secondary structure that includes two domains. Each domain consists of two short base paired helices separated by an internal loop.
- Domain A (Helix 1- loop A-Helix 2) contains substrate and primary substrate-recognition region of ribozyme. Domain B (Helix 3- loop B-Helix 4) contains primary catalytic determinants of ribozyme.
- Both domains are joined covalently via a phosphodiester linkage, connecting Helix 2 of Domain A to Helix 3 of Domain B. This linkage helps in initiating the catalysis process.
Key Points to Remember
Here is the list of key points we need to remember about “Introns, Exons, and Ribozymes”.
- Exons are coding sequences expressed in mRNA, while introns are non-coding regions removed during RNA processing.
- RNA splicing joins exons and removes introns; Group I introns self-splice, whereas Group II and III introns require the spliceosome.
- Exon shuffling rearranges exons from different genes, promoting genetic diversity and new protein functions.
- Ribozymes are RNA molecules that catalyze reactions and can self-splice, playing vital roles in cellular processes.
- Hammerhead ribozymes cleave RNA without proteins and are consumed in the reaction, while hairpin ribozymes use a specific structure for catalysis and don’t require metal ions.