Review_ch._14_-_17-2

Page 1: DNA Structure and Replication

• Nucleotide Polymers: DNA and RNA are made of nucleotides, with DNA containing thymine (T) and RNA containing uracil (U).

• Phosphodiester Bond: Nucleotides connect through phosphodiester bonds, forming a backbone in a 5’ to 3’ orientation, adhering to Chargaff's rules.

• Discovery of DNA Structure:

  • Rosalind Franklin provided DNA's crystal structure.

  • Watson and Crick proposed the double helix model of DNA.

  • DNA features a major and minor groove, with hydrogen-bond pairing: A:T (2 H-bonds) and G:C (3 H-bonds).

• Antiparallel Configuration:

  • One DNA strand ends in a 3’ hydroxyl (-OH) and the other in a 5’ phosphate.

• DNA Replication Process:

  • Requirements: DNA template, enzymes, and nucleotide triphosphates.

  • Stages: 1) Initiation, 2) Elongation (synthesis of new DNA strands), 3) Termination.

• Role of DNA Polymerases:

  • DNA polymerase III attaches a nucleotide monophosphate from dNTPs, releasing pyrophosphate.

  • Requires RNA primer, synthesizes in the 5’ to 3’ direction.

  • E. coli has three polymerases: I (lagging strand primer), II (repair), III (main replication).

• Proofreading and Repair:

  • All polymerases have 3’ to 5’ exonuclease (proofreading) capabilities.

  • Pol I also has a 5’ to 3’ exonuclease to remove primers.

• Unwinding DNA:

  • Helicases unwind DNA using ATP; Single-Stranded Binding (SSB) proteins stabilize separated strands, while DNA gyrase (topoisomerase) prevents supercoiling.

• Semi-discontinuous Replication:

  • Leading Strand: synthesized continuously.

  • Lagging Strand: synthesized in Okazaki fragments with RNA primers made by Primase.

• Assembly of Replication Enzymes:

  • Replisome consists of primase, helicase, accessory proteins, and two DNA Pol III.

  • Gyrase reduces DNA strain, and helicase separates strands.

• Replication Fork Mechanics:

  • Primase synthesizes primers for lagging strands which form loops enabling 5’ to 3’ synthesis.

  • DNA polymerase I replaces RNA primers with DNA and DNA ligase joins the fragments.

• Termination:

  • Termination occurs at specific sites, with gyrase unlinking DNA copies.

Page 2: Eukaryotic Replication and DNA Repair

• Eukaryotic vs Prokaryotic Replication:

  • Eukaryotes have multiple origins of replication (replicons); prokaryotes have one origin.

  • Eukaryotic priming involves Pol α and primase, while Pol ε and δ are for leading and lagging strands respectively.

• Telomeres and Protection:

  • Telomeres protect chromosome ends; synthesized by telomerase using an RNA template.

  • Telomerase is active during childhood and low in adults, except in dividing cells (e.g., lymphocytes, cancer).

• DNA Repair Mechanisms:

  • Mismatch Repair (MMR): corrects incorrect bases.

  • Photo Repair: uses photolyase for thymine dimers.

  • Excision Repair: replaces damaged regions using undamaged strands as templates.

• Historical Research on Genes:

  • Garrod identified alkaptonuria as a recessive trait affecting enzymes.

  • Beadle and Tatum's experiment with bread mold concluded that one gene corresponds to one enzyme (1-gene/1-polypeptide hypothesis).

• Central Dogma of Molecular Biology:

  • Flow: DNA (transcription) → RNA (translation) → Protein.

  • Retroviruses reverse this flow (RNA → DNA).

• RNA Synthesis:

  • The template strand of DNA is transcribed into RNA, replacing T with U.

  • mRNA directs polypeptide synthesis; rRNA is necessary for protein synthesis; tRNA carries amino acids.

  • Small RNAs (miRNA, siRNA) control gene expression.

Page 3: Genetic Code and Transcription

• Genetic Code Basics:

  • Codons: sequences of 3 nucleotides that correspond to amino acids.

  • Key codons: Start (AUG), Stop (UAA, UGA, UAG).

  • Degeneracy: multiple codons can specify the same amino acid.

• RNA Polymerase Functions:

  • Prokaryotes have a single RNA polymerase (holoenzyme).

  • Transcription requires a promoter for initiation and terminator for ending, with no need for a primer.

• Transcription Phases:

  • Initiation: σ subunit binds DNA; polymerase attaches and σ dissociates.

  • Elongation: RNA chain grows in the 5’ to 3’ direction.

  • Termination: hairpin formation and weak bonds lead to disassociation of RNA.

• Transcription Coupling:

  • In prokaryotes, transcription occurs simultaneously with translation.

  • Operons organize genes into transcriptional units controlled together.

• Eukaryotic Transcription:

  • Eukaryotes have three types of RNA polymerases (I, II, III) for rRNA, mRNA, and tRNA synthesis respectively.

  • Eukaryotic promoter complexity requires transcription factors for initiation.

• Post-transcriptional Modifications:

  • Primary mRNA transcripts require 5’ GTP cap, 3’ poly-A tail, and splicing of introns to form mature mRNA.

• Splicing Mechanism:

  • snRNPs recognize intron boundaries, forming a spliceosome to splice out introns.

  • Exons are joined together after lariat formation.

Page 4: Transcriptome and Proteome

• Understanding Transcriptome and Proteome:

  • Transcriptome: all RNA from a genome; Proteome: all proteins expressed.

  • The ratio of genes to transcripts to proteins is not uniform due to alternative splicing.

• Translation Mechanism:

  • Ribosomes require mRNA and tRNA for protein synthesis.

  • tRNA holds anticodons complementary to mRNA codons.

  • Aminoacyl-tRNA synthetases attach amino acids to tRNA.

• Ribosomal Structure:

  • Ribosomes have two subunits with three binding sites (A, P, E).

  • Peptide bonds formed by peptidyl transferase in the ribosomal large subunit.

• Translation Initiation and Elongation:

  • Prokaryotic initiation includes the small subunit, mRNA, and fMet-tRNA.

  • In eukaryotic initiation, the initiator is Met, with a more complex assembly.

  • Elongation involves tRNA binding to A site and peptide bond formation, followed by translocation.

• Termination of Translation:

  • Recognition of stop codons by release factors signals termination; completed polypeptide is released.

• Gene Mutation and Repair:

  • Mutation is a heritable alteration; point mutations can be synonymous, missense, or nonsense.

  • Indels and frameshift mutations alter reading frames.

  • Trinucleotide repeat expansions, such as in Huntington's disease, lead to neurodegenerative disorders.

• Mutation Types and Rates:

  • Structural variants affect larger segments; human mutation rates are documented with significant interest in understanding genetic diversity.

Page 5: Gene Expression Control

• Regulation of Gene Expression:

  • Gene expression regulated at the transcription initiation level; prokaryotes respond to environmental changes, while eukaryotes maintain homeostasis.

• Regulatory Proteins:

  • Bind DNA to influence RNA polymerase activity: can either block (negative control) or activate (positive control) transcription.

• Prokaryotic Operons:

  • Genes functionally related are organized in operons; e.g., Lac operon for lactose metabolism.

  • Repressors and effectors modulate expression based on substrate availability (e.g., glucose vs. lactose).

• Tryptophan Operon:

  • Regulates the synthesis of trp in response to amino acid availability.

Page 6: Eukaryotic Transcription Factors

• Complexities in Eukaryotic Regulation:

  • Eukaryotes use specific transcription factors that require interactions with general factors for initiation of transcription.

  • Enhancers can influence transcription over large distances by altering DNA structure.

• Chromatin and Epigenetics:

  • Chromatin structure influences accessibility; epigenetic changes can affect gene expression without altering DNA sequences.

  • Histone modifications, such as acetylation, methylation, and phosphorylation, affect chromatin compaction.

• Post-Transcriptional Regulation:

  • miRNAs and siRNAs play critical roles in fine-tuning gene expression through RNA silencing pathways, affecting target mRNA stability and translation efficiency.

Page 7: Advanced Regulation and Mutations

• Tissue-Specific Expression:

  • Alternative splicing generates diverse protein isoforms from the same gene; specific isoforms can have distinct functions in different tissues.

• Regulation by RNA Editing:

  • Editing alters base pairing, changing how mRNAs are translated into proteins, providing variability in protein function.

• Proteasomal Degradation:

  • Ubiquitin tags proteins for degradation, maintaining protein balance within cells.

• Recombinant DNA Technology:

  • Techniques such as PCR allow for the amplification and manipulation of DNA sequences.

Page 8: Biotech and Gene Editing

• CRISPR-Cas9 System:

  • A revolutionary genome editing tool that uses guide RNAs to direct CAS9 for cutting DNA at specific locations, allowing for gene manipulation and analysis.

• Applications of Recombinant DNA:

  • Production of therapeutic proteins and genetically modified organisms for agricultural enhancement.

• Analysis Techniques:

  • FISH and DNA microarrays are powerful techniques for identifying genetic disorders and studying gene expression.

Page 9: Conclusion

• Integration of Molecular Biology Techniques:

  • The advances in molecular biology provide powerful tools for genetic research and biotechnology applications, reshaping our understanding of genetics and enhancing medical and agricultural fields.