Lecture 2
BM210: Introduction to Biochemistry Lecture 2: Eukaryotic Gene Expression and Function
Learning Outcomes
Definitions of Key Terms: Key terms related to eukaryotic gene expression, including:
Introns: Non-coding sequences within a gene.
Exons: Coding sequences that remain in the RNA after splicing.
Spliceosome: A complex responsible for RNA splicing.
Codons: Sequences of three nucleotides in mRNA that correspond to specific amino acids.
Ribosomes: Molecular machines that synthesize proteins based on the mRNA template.
Differentiation of Splicing Types: Distinguish between constitutive splicing and alternative splicing, and identify how these processes contribute to protein diversity.
Assessment of Mutations: Evaluate the impact of mutations on RNA splicing, discussing how changes can lead to diseases.
Significance of Catalytic RNA: Understand the historical significance of catalytic RNA in molecular biology and its applications in biotechnology.
Translation Steps Overview: Examine the steps involved in translation, particularly the roles of tRNA, aminoacyl-tRNAs, ribosomes, and the genetic code.
mRNA Splicing
Genes as Organizing Units of DNA
Average human gene:
Contains approximately 8 introns.
Some genes can have more than 100 introns.
Intron sizes range from 50 to 10,000 nucleotides (10 kb).
Experiments in mRNA Splicing
Experiment Overview:
Used hybridization of purified hexon mRNA and transcribed strand from Ad2 EcoRI A DNA fragment.
Diagram Depiction:
Shows hexon mRNA exons (red) and introns (light blue among A, B, and C) across a 25 kb segment of the Ad2 genome.
Significance:
Revealed that mRNA contains both coding and non-coding regions.
Discovery of Introns
The discovery of mRNA splicing was conducted by Richard J. Roberts and Phillip A. Sharp in the late 1970s.
Findings:
Genes contain introns that are spliced out during mRNA processing, leading to a new understanding of gene structure, termed split genes.
Recognition: Both awarded the Nobel Prize in Physiology or Medicine in 1993 for their groundbreaking work.
Introns: Functions and Characteristics
Definition: Introns are noncoding DNA sequences located between exons.
Characteristics:
Number and length increase with organism complexity.
Functions:
Facilitate alternative splicing.
Participate in gene expression regulation.
Contribute to evolution and protein diversity.
Involved in mRNA transport, chromatin assembly, and nonsense-mediated decay.
Provide a buffering function for genetic changes.
Processing of Pre-mRNA
Pre-mRNA Characteristics:
Synthesized by RNA polymerase II.
Known as unspliced mRNA, is unstable and must be processed to prevent degradation.
Processing Steps:
Addition of a 5’ cap.
Addition of a 3’ poly(A) tail.
Intron splicing occurs following these modifications.
Intron Splicing Overview
Definition: Splicing is the process of removing introns and ligating exons together to form a mature mRNA transcript.
Lariat Formation: The excised intron is known as a lariat due to its looped structure.
Mechanism of Intron Splicing
Recognition Elements: Sequencing indicates all introns start with GU and end with AG.
They contain a pyrimidine-rich tract (Py)n and a specific adenine, termed the branch site.
Pyrimidines: The nucleotides involved are C, T, and U.
Role of Small Nuclear RNAs and Proteins in Splicing
snRNAs: Small nuclear RNAs (average fewer than 300 nucleotides) catalyze splicing by forming complexes called small nuclear ribonucleoproteins (snRNPs) or “snurps”.
Functionality: These snRNPs detect specific splice sites and recruit other snRNPs to form the spliceosome.
Splicing Complex Formation: The spliceosome captures, splices, and releases RNA in a coordinated manner.
Note: The splicing mechanism intricacies are visually supported by a linked video demonstrating the spliceosome's function.
Detailed Splicing Mechanism
Activation: Begins with the cleavage of the phosphodiester bond between exon 1 and the 5' end of the intron.
Attack: The 2’-OH group of an adenylate residue at the branch site performs a nucleophilic attack causing the formation of a 2’-to-5’ phosphodiester bond via transesterification.
Formation of Peptide Bond: Subsequent to forming the bond with the 5' terminal phosphate of the intron, the 3’ OH group of exon 1 attacks the intron-exon 2 bond, resulting in exon 1 and exon 2 joining, thereby releasing the intron as a lariat.
Types of Splicing
Constitutive Splicing
Definition: Involves removal of all introns and joining of all exons in a predetermined order, resulting in a single, uniform mRNA transcript.
Only 5% of genes in the human genome undergo constitutive splicing.
Alternative Splicing
Definition: Alternative splicing is a process where different mRNAs can be produced from the same primary transcript due to the inclusion or exclusion of certain exons.
Significance:
Allows for protein diversity by generating proteins with varied functions from one gene.
Essential for regulating gene expression and function.
Example of Potential Variants:
A gene with 6 exons and 5 introns can produce 16 different mRNAs and additional proteins besides the full-length protein.
Case Study: The calcitonin gene (CALCA) generates:
Calcitonin – hormone for calcium and phosphorus metabolism in thyroid cells.
Calcitonin Gene-Related Peptide (CGRP) – functions as a vasodilator and neuromodulator in neuronal cells.
Katacalcin – also generated in thyroid cells, has a role in calcium metabolism.
Impact of Alternative Splicing on Protein Diversity
Statistical Insight: Sequencing reveals that most pre-mRNAs are alternatively spliced, leading to increased protein diversity.
Alternative splicing can produce over 90,000 proteins from merely 25,000 genes in humans, suggesting a greater than 4-fold increase in proteins than genes.
Control Mechanisms of Alternative Splicing
Cis-acting Elements: Specific sequences within the pre-mRNA help to recognize exons and influence splicing outcomes.
Trans-acting Factors: Proteins that bind to cis elements, notably SR proteins which are concentrated in nuclear granules and affect splicing dynamics via phosphorylation.
Nuclear Speckles: Regions in the nucleus where splicing factors accumulate, enhancing interaction with nearby genes.
Consequences of Splicing Mutations
Mutations in splicing can lead to diseases by disrupting normal mRNA processing.
Self-Splicing Mechanisms
Some RNA can splice itself due to specific folding patterns that create structures necessary for the splicing reaction.
Translation: Protein Synthesis
Overview of Translation Process
Role of mRNA: Acts as a template for protein synthesis where specific amino acids are decoded from the genetic information.
Function of tRNAs: Translate genetic codes, facilitating the addition of amino acids to the growing polypeptide chain.
Location: Translation occurs within the cytosol on ribosomes.
The Genetic Code
Definition: The genetic code corresponds to a sequence of nucleotides that guide protein synthesis via codons (combinations of three nucleotides).
Codon Functionality: Each codon specifies an amino acid; different codons may correspond to the same amino acid.
Representation: Amino acids can be denoted using either a three-letter or a one-letter code.
Start and Stop Codons:
Translation initiates at the AUG codon (Methionine) and concludes at UAA, UAG, or UGA (stop codons).
Untranslated Regions (UTRs)
The 5' UTR is an area of mRNA that does not code for amino acids; it is crucial for processing and translation initiation but does not influence the protein sequence.
Transfer RNA (tRNA)
tRNA has a cloverleaf secondary structure stabilized by hydrogen bonds among complementary bases.
At one end, each tRNA possesses an amino acid attachment site and at the opposite end an anticodon, which pairs with the respective codon in mRNA.
Aminoacyl-tRNA Synthetases
Enzymes that catalyze the formation of aminoacyl-tRNAs by linking specific amino acids to their corresponding tRNAs with the help of ATP.
Three-step reaction:
Amino acid + ATP + tRNA + H2O → Aminoacyl-tRNA + AMP + 2 Pi
Ribosome Structure and Function
Comprised of two subunits: large (60S) and small (40S), forming an 80S ribosome.
Ribosomes contain three binding sites:
A site: Where aminoacyl-tRNA base pairs with mRNA codon.
P site: Contains the tRNA associated with the growing polypeptide.
E site: Where the uncharged tRNA resides before release.
Translation Initiation
Formation of complex with small ribosomal subunit, initiation tRNA (Met), initiation factors, and GTP, binding at mRNA cap and scanning for the AUG codon.
Once found, the large subunit and elongation factors are recruited, initiating protein synthesis.
Translation Elongation
The ribosome translocates along mRNA in the 5'-to-3' direction, moving first tRNA from A to P and releasing it from the E site.
New tRNA corresponding to the next codon occupies the A site, requiring elongation factors and GTP.
Peptidyl transferase activity forms peptide bonds between adjacent amino acids, proceeding until reaching a stop codon.
Translation Termination
Recognized by the three stop codons (UAA, UAG, UGA), which do not bind with tRNAs.
Release Factors (RF) bind to stop codons at A site, promoting cleavage of the polypeptide chain and disassembly of the ribosome.
Summary of Splicing and Translation
RNA Modifications: Importance of the 5’ cap and poly-A tail in mRNA stability and translation.
Key Definitions: Introns, exons, spliceosome, codons, ribosomes, and aminoacyl-tRNA synthetases.
Main Steps of RNA Processing: Including capping, polyadenylation, and splicing, followed by translation.
Mechanism of mRNA Splicing: Recognition of splice sites and formation of spliceosome.
Constitutive vs Alternative Splicing: How alternative splicing contributes to protein diversity.
Understanding the Genetic Code: Its role in protein synthesis.
Implications of Mutations: Their effect on RNA splicing and translation, potentially leading to disease.
Applications: Insights into splicing and translation mechanisms for therapeutic and biotechnological developments.