Non-Coding DNA & Gene Structure - PDF

1. Non-Coding DNA Sequences: Non-coding DNA refers to regions in the genome that do not code for proteins. While protein-coding genes make up only about 1.5% of the human genome, non-coding sequences serve several important functions. Functions of Non-Coding Sequences: ○ Sa...

1. Non-Coding DNA Sequences: Non-coding DNA refers to regions in the genome that do not code for proteins. While protein-coding genes make up only about 1.5% of the human genome, non-coding sequences serve several important functions. Functions of Non-Coding Sequences: ○ Satellite DNA (e.g., short tandem repeats) is used in DNA profiling to differentiate individuals. ○ Telomeres are repetitive sequences at the ends of chromosomes that protect against chromosomal deterioration during replication. ○ Introns are non-coding regions within eukaryotic genes that are removed during RNA processing. ○ Non-coding genes (e.g., tRNA, rRNA) produce functional RNA that does not code for proteins. ○ Gene regulatory sequences (e.g., enhancers and silencers) regulate transcription by affecting RNA polymerase binding. 2. Gene Structure: A gene consists of three main sections: promoter, coding sequence, and terminator. 3. Promoter: The promoter is a non-coding sequence upstream of a gene that initiates transcription. It serves as a binding site for RNA polymerase and is regulated by transcription factors. ○ Transcription factors can either activate or repress the binding of RNA polymerase, regulating gene expression. 4. Coding Sequence: The coding sequence is the part of the gene that is transcribed into RNA. The RNA transcript is complementary to the coding sequence, except that uracil (U) replaces thymine (T). 5. Terminator:  The terminator is the sequence that signals the end of transcription. In both prokaryotes and eukaryotes, the mechanism of transcriptional termination differs. 6. Directionality of Transcription: Transcription occurs in a 5’ → 3’ direction, where RNA polymerase adds RNA nucleotides to the 3’ end of the growing mRNA strand. This directionality is determined by the orientation of the DNA and the action of RNA polymerase. Key Takeaways: Non-coding DNA plays essential roles in gene regulation, chromosomal protection, and RNA production, even though it doesn’t directly code for proteins. A gene has three main parts: promoter (where transcription starts), coding sequence (which is transcribed into RNA), and terminator (which signals the end of transcription). Transcription is driven by RNA polymerase, and occurs in the 5’ → 3’ direction, synthesizing mRNA complementary to the DNA template. 1. Post-Transcriptional Modifications in Eukaryotes: Eukaryotic cells undergo three major post-transcriptional events to produce mature mRNA: ○ Capping ○ Polyadenylation ○ Splicing These processes do not occur in prokaryotes, as their lack of compartmentalization and more compact genomes prevent such modifications. 2. Capping: Capping adds a methyl group to the 5’-end of the RNA transcript. This methylated cap: ○ Protects the RNA from degradation by exonucleases. ○ Helps the RNA to be recognized by the translational machinery (e.g., ribosome and nuclear export proteins). 3. Polyadenylation: Polyadenylation involves the addition of a poly-A tail (a long chain of adenine nucleotides) to the 3’-end of the RNA transcript. The poly-A tail: ○ Increases RNA stability. ○ Aids in the export of the RNA from the nucleus to the cytoplasm. 4. Splicing: Splicing removes introns (non-coding sequences) from the RNA transcript and joins the exons (coding regions). The resulting RNA is a continuous sequence of exons, forming the mature mRNA. Alternative splicing can occur, where: ○ Different combinations of exons are joined together to produce protein variants from a single gene. ○ This allows the same gene to produce multiple polypeptides, such as producing a protein that can either be membrane-bound or cytosolic, depending on the presence of specific exons. Key Takeaways: Capping, polyadenylation, and splicing are essential for converting the primary RNA transcript into mature, functional mRNA in eukaryotes. Capping and polyadenylation help stabilize and protect the mRNA, while splicing removes non-coding sequences (introns) and can also generate different protein variants through alternative splicing. 1. Chemical Diversity in Amino Acids: Proteins are made up of 20 common amino acids, each having a distinctive side chain (R-group). The chemical properties of the side chains (hydrophobic, hydrophilic, polar, charged, acidic, or basic) determine how the protein folds and functions. This diversity allows for a wide range of protein shapes and functions, enabling organisms to produce a vast variety of polypeptides with specialized roles. 2. Key Steps in Translation (Polypeptide Synthesis):  Translation has four main stages: initiation, elongation, translocation, and termination. 3. Initiation: The small ribosomal subunit binds to an initiator tRNA that recognizes the start codon (AUG). The complex attaches to the 5’-end of the mRNA and moves towards the start codon. The large ribosomal subunit binds at the start codon to form the complete ribosomal complex, and initiation factors are released. 4. Elongation: The ribosome has three tRNA binding sites: A site (aminoacyl), P site (peptidyl), and E site (exit). The initiator tRNA binds to the P site, and a second tRNA enters the A site, bringing the next amino acid. A peptide bond forms between the amino acids in the P and A sites, and the tRNA in the P site is deacylated (loses its amino acid). 5. Translocation: The ribosome moves along the mRNA by one codon (5’ → 3’), shifting the tRNAs: ○ The tRNA in the P site moves to the E site and is released. ○ The tRNA carrying the growing peptide chain moves to the P site. ○ The cycle repeats with a new tRNA entering the A site. 6. Termination: Elongation and translocation continue until a stop codon is encountered. Release factors bind to the stop codon, triggering the release of the polypeptide chain and the disassembly of the ribosome. Key Takeaways: The chemical properties of amino acid side chains are crucial for the diversity of protein structure and function.  The process of translation involves initiation, elongation, translocation, and termination, where tRNAs play a key role in adding amino acids to the growing polypeptide chain, and ribosomes move along the mRNA to synthesize proteins. 1. Primary Structure (1º): Primary structure refers to the sequence of amino acids in the polypeptide chain, determined by covalent peptide bonds between adjacent amino acids. This sequence controls all subsequent protein structures (secondary, tertiary, and quaternary) because it dictates the interactions between the R groups of amino acids, which influence folding and shape. 2. Secondary Structure (2º): The secondary structure involves the folding of the polypeptide chain into specific shapes, such as α-helices(spirals) and β-pleated sheets (arrows), stabilized by hydrogen bonds between the amine and carboxyl groups of non-adjacent amino acids. These structures provide mechanical stability to the polypeptide chain. 3. Tertiary Structure (3º): The tertiary structure is the 3D shape of the protein, formed by interactions between the R groups of amino acids. Key interactions include: ○ Hydrogen bonds, ○ Ionic bonds, ○ Disulfide bridges (covalent bonds between sulfur atoms in cysteine), ○ Hydrophobic interactions (non-polar amino acids avoiding water). The tertiary structure is critical for the protein’s function, especially in enzymes where the specificity of the active site depends on its precise 3D shape. 4. Quaternary Structure (4º): The quaternary structure occurs in proteins with more than one polypeptide chain (e.g., insulin and collagen). Some proteins have a conjugated quaternary structure, where inorganic prosthetic groups (like heme in hemoglobin) are added to the protein.  Not all proteins have a quaternary structure; many proteins are monomeric (single polypeptide chain). Key Takeaways: Primary structure (amino acid sequence) sets the foundation for the protein's overall structure. The secondary structure involves hydrogen bonds and leads to regular folding patterns (α-helices and β-pleated sheets). The tertiary structure is a more complex 3D fold stabilized by various interactions between the side chains (R groups). The quaternary structure involves multiple polypeptide chains and is essential for proteins with multiple subunits or additional functional groups (e.g., heme in hemoglobin). 1. Globular Proteins: Shape and Solubility: Globular proteins are spherical and water-soluble. Function: They typically perform biological activities, such as acting as enzymes, hormones, or antibodies. Structure: Globular proteins often have an irregular amino acid sequence, and their folding allows them to carry out specific functions. Example: Insulin is a globular protein that regulates blood sugar. Its structure includes two polypeptide chainsconnected by disulfide bridges, with a hydrophilic exterior allowing it to travel freely in the bloodstream. 2. Fibrous Proteins: Shape and Solubility: Fibrous proteins are long, narrow, and insoluble in water. Function: They mainly have structural roles, helping to provide strength and stability (e.g., in connective tissues, bones, and skin). Structure: They have a repetitive amino acid sequence, often forming helical shapes or cross-linked structures(e.g., collagen). Example: Collagen is a fibrous protein made of three polypeptide chains in a triple helix. The hydrophobic exterior helps it remain stable in aqueous environments, contributing to its structural role in tissues. 3. Effect of Amino Acid Polarity on Tertiary Structure: Polar vs Non-Polar Amino Acids: ○ Polar (hydrophilic) amino acids are typically found on the surface of proteins to interact with water. ○ Non-polar (hydrophobic) amino acids tend to be found in the interior to provide stability. Protein Folding: The distribution of polar and non-polar amino acids influences how the protein folds and how it interacts with its environment. Example in Membrane Proteins: Integral membrane proteins have non-polar amino acids on their surface (to interact with the lipid membrane), while polar amino acids form hydrophilic channels within the protein. Key Takeaways: Globular proteins are functional, spherical, and soluble, performing tasks like enzyme catalysis and hormonal regulation (e.g., insulin). Fibrous proteins are structural, long, and insoluble, contributing to mechanical strength (e.g., collagen). The polarity of amino acids in a protein determines its folding, solubility, and function, influencing how it interacts with water or cell membranes. 1. Post-Translational Modifications: Definition: After translation, some proteins need modifications to become functional. Types of Modifications: ○ Disulfide bridges: Covalent bonds formed between cysteine residues, stabilizing the protein's structure. ○ Conjugation with cofactors: Proteins may bind with other molecules (e.g., haeme group in hemoglobin) to function properly. ○ Chemical modifications: Processes like glycosylation (adding sugars) or phosphorylation (adding phosphate groups) can affect stability and biological activity. ○ Proteolytic cleavage: Removal of amino acids from the polypeptide chain, aiding in protein folding or activation. 2. Insulin Modification Example:  Inactive Precursor: Preproinsulin is synthesized as an inactive form in the rough endoplasmic reticulum. First Step: The signal sequence is removed, converting preproinsulin into proinsulin. Second Step: In the Golgi complex, disulfide bridges form between the A and B chains, and the C peptide is removed. The mature insulin is then packaged into secretory vesicles for regulated secretion. Key Takeaways: Post-translational modifications are crucial for protein functionality, involving processes like disulfide bond formation, cofactor binding, chemical modifications, and cleavage of the protein chain. Insulin is a prime example of how a protein undergoes sequential modifications (e.g., signal sequence removaland disulfide bond formation) to become biologically active and functional. 1. Proteasome Function: Proteasomes are large protein complexes responsible for degrading unneeded or misfolded proteins. They break peptide bonds in proteins, a process called proteolysis, and recycle the amino acids for future protein synthesis. 2. Ubiquitination: Ubiquitin is a small protein that tags proteins for degradation. Proteins targeted for destruction are ubiquitinated, marking them for recognition by the proteasome. 3. Proteasome Structure and Mechanism: Ubiquitinated proteins are recognized by the outer regulatory complex of the proteasome, which facilitates their entry into the proteolytic core. The proteasome has an ⍺-subunit gate that controls access to the core, preventing unnecessary proteolysis. 4. Role in Protein Regulation:  The proteasome plays a key role in regulating protein expression levels and helps maintain the functional proteome by controlling protein degradation and recycling amino acids. Key Takeaways: Proteasomes degrade unwanted proteins and recycle their amino acids, ensuring proper protein turnover. The process starts with ubiquitination, marking proteins for degradation, and ends with the recycling of amino acids to maintain cellular function.