Central Dogma of Molecular Biology Chapter 24

Central Dogma of Molecular Biology

• The central dogma describes how genetic information moves from DNA to proteins
• Genetic information in DNA cannot be reversed once it is translated into proteins
• Replication: DNA is replicated into an identical copy of DNA
• Transcription: DNA is transcribed into RNA (via mRNA)
• Translation: RNA is translated into proteins

Genes and Genomes

• Genes: segments of DNA that code for RNA or proteins
• Genomes: complete set of genetic information in an organism
• Genes can code for different products
• Eukaryotic DNA is non-coding, leading to complexity not being related to the number of chromosomes or DNA length

Eukaryotes and Prokaryotes

• Compare:
  • Both undergo transcription and translation for replication
• Contrast:
  • Eukaryotes: diploid, linear DNA, telomeres, introns, histones
  • Prokaryotes: single chromosome, circular DNA, no telomeres, no introns

DNA Organization

• Cytosol: observed in prokaryotes, contains nucleoid with DNA
• Nucleus: observed in eukaryotes, contains DNA arranged into chromosomes
• Nucleoids: observed in prokaryotes, contains circular bacterial DNA
• Mitochondria: contains double-stranded circular DNA encoding mitochondrial tRNA, rRNA, and proteins
• Chloroplast: contains double-stranded circular DNA encoding proteins

DNA Structure

• DNA: deoxyribonucleic acid containing genetic information
• Chromosome: large molecule composed of DNA and proteins
• Genes: sequences of DNA or RNA coding for particular functions
• Centromeres: regions holding daughter chromosomes during mitosis
• Telomeres: regions ending eukaryotic chromosomes

Human Genome

• Contains a small number of genes encoding proteins (exons)
• Non-coding DNA: introns, promoters, terminators, and regulatory sequences
• Half of the human genome is repetitive, with sequences like transposons

DNA Supercoiling

• Level of organization allowing DNA packing in cells
• Influences transcription and replication
• DNA is most stable in the B-form structure
• Twist, writhe, and linking number are important for DNA structure and function

Topoisomerases

• Enzymes catalyzing changes in the linking number
• Type I: removes negative supercoils, relaxes DNA
• Type II: relaxes negative and positive supercoils, introduces negative supercoils in bacteria

Clinical Correlation

• Antibiotics: inhibit topoisomerase function in bacteria
• Chemotherapy: targets topoisomerase in cancer cells

Cell Cycle

• Goes through Go, G1, S, G2, and M phases
• Chromatin is undefined and dispersed in interphase
• Chromosomes condense and become visible during mitosis

DNA Packing

• Process of tightly packing DNA strands into the nucleus
• DNA is wrapped around histones, forming nucleosomes
• Nucleosomes are coiled into chromatin fibers

Chromosome Structure

• Cohesins: connect sister chromatids after replication
• Condensins: condense chromosomes during mitosis
• Positive supercoils are formed during chromosome condensation

DNA Replication

• Semi-conservative, using each DNA strand as a template
• Bidirectional, proceeding in both directions from the origin
• Discontinuous synthesis occurs in the lagging strand
• Okazaki fragments are short pieces of DNA synthesized in the lagging strand

Meselson-Stahl Experiment

• Demonstrated semi-conservative replication of DNA
• Used heavy nitrogen to label DNA

DNA Replication Terms

• Leading strand: synthesized continuously in the 5’ to 3’ direction
• Lagging strand: synthesized discontinuously in the 5’ to 3’ direction
• Continuous 5’-3’ synthesis: occurs in the leading strand
• Discontinuous 5’-3’ synthesis: occurs in the lagging strand
• Okazaki fragments: short pieces of DNA synthesized in the lagging strand
• DNA polymerase: adds nucleotides to the 3’ end of the DNA strand
• Primase: synthesizes RNA primers
• Ligase: seals the gaps between Okazaki fragments

Replication Bubble

• Direction of synthesis: 5’ to 3’
• Action of DNA polymerase: adds nucleotides to the 3’ end
• Requirements of single-strand binding proteins: stabilize single-stranded DNA
• Action of helicase: unwinds the DNA double helix
• Action of topoisomerase: relieves stress caused by DNA unwinding

DNA Replication Rules

• Semi-conservative replication
• Replication begins at a specific origin
• Replication proceeds bidirectionally
• Replication is semi-discontinuous

Prokaryotic and Eukaryotic DNA Replication

• Similarities:
  • Both use DNA polymerase with 3’ to 5’ exonuclease activity
  • Both use primers that are removed by RNase
• Differences:
  • Prokaryotes: occurs in the cytoplasm, less complex, uses DNA gyrase, and has a single origin
  • Eukaryotes: occurs in the nucleus, more complex, regulated by cyclins and cyclin-dependent kinases, and has multiple origins### RNA Synthesis (Transcription)
• Template Strand: Used as a template for RNA polymerase to act on, moves in a 3' to 5' direction
• Coding Strand: Non-template strand with the same base sequence as the RNA transcribed, moves in a 5' to 3' direction, T is replaced with U
• Promoter Sequence: DNA sequence that RNA polymerases bind to, directs the direction of transcription
• Primary Transcript (hnRNA): Newly synthesized RNA molecules
• Upstream/Downstream: Upstream located at the beginning of transcription, containing regulatory proteins; downstream located after the start of transcription

Transcription Signals

• Proximal Elements: Transcription signals found at the start of transcription
• Distal Elements: Transcription signals found downstream
• Cis-acting Elements: Binding sites for promoters, enhancers, and silencers, located in the same DNA molecule they regulate
• Trans-acting Elements: Bind to cis-acting elements to regulate gene expression, regulate genes in different DNA molecules
• Enhancer and Silencer Elements: Enhancers increase the rate of transcription, silencers decrease the rate of transcription

Eukaryotic RNA Polymerases

• RNA Pol I, II, and III: Each has a regulation mechanism
• RNA Pol I: Responsible for synthesizing pre-ribosomal RNA
• RNA Pol II: Responsible for synthesizing mRNA, fast and can recognize many promoters, inhibited by α-amanitin
• RNA Pol III: Responsible for synthesizing tRNAs and small RNA products
• RNA Pol IV: Used in plants to synthesize small interfering RNAs (siRNAs)
• Mitochondrial RNA Polymerase: Used in mitochondria

Maturation of mRNA

• Synthesis of hnRNA: Also called pre-mRNA, transcribed in the nucleus by RNA pol II
• 5'-Capping: 7-methylguanosine added to the 5' end of RNA to protect from ribonuclease
• Poly-A Tail Addition: Poly-adenylation added to the 3' end to make mRNA stable
• Intron Removal by Spliceosome: Introns removed from mRNA by spliceosome

Intron Classes and Splicing

• Group I and II Introns: Self-splicing, found in genomes of nuclear, mitochondrial, and chloroplast
• Spliceosomal Introns: Most common introns, spliced by spliceosomes
• tRNA Introns: Protein-catalyzed, spliced by ligase and splicing endonuclease

Pre-mRNA Splicing Defects and Disease

• Alternative Splicing: Defect can cause spinal muscle atrophy

Eukaryotic RNA Species and Maturation

• mRNA: Made in the nucleus via RNA pol II, matures through 5'-capping, poly-A tail addition, and splicing
• rRNA: Made in the nucleus via RNA pol I, matures through processing and modification
• tRNA: Made in the nucleus via RNA pol II, matures through processing and modification

Base Modification in tRNA and rRNA

• Pseudouridine, Thiouridine, and Dihydrouridine: Post-transcriptional modifications in tRNA and rRNA

MicroRNAs and RNA Interference

• MicroRNAs (miRNAs): Short, noncoding nucleotides that bind to specific regions of mRNA to affect translation
• RNA Interference (RNAi): Mechanism for processing miRNAs to silence gene expression

Ribozymes, Retroviruses, and Reverse Transcriptase

• Ribozymes: RNA molecules that catalytically cleave themselves or other RNA
• Retroviruses: RNA viruses that can convert RNA to DNA via reverse transcriptase
• Reverse Transcriptase: Enzyme that converts RNA to DNA in retroviruses

Telomeres and Telomerases

• Telomeres: Region at the end of eukaryotic chromosomes, maintained by telomerase
• Telomerase: Enzyme that extends the ends of chromosomes, serves as a reverse transcriptase

Genetic Information Flow

• Universal Flow: Genetic information in DNA is replicated and transcribed into RNA, then translated into proteins
• Exceptions: Reverse transcription, RNA viruses, Prions, and RNA editing

Genetic Code

• Degenerate: Multiple codons can code for the same amino acid

• Unambiguous: A specific codon only codes for a specific amino acid

• Nonoverlapping: Each nucleotide base is only part of one codon

• Universal: Codons are the same across most species### Regulation of Gene Expression

• Repression: a process where gene expression decreases in response to a change in a regulatory protein.

• Positive Control: induces transcription through protein activators, allowing for signals to bind to DNA and induce transcription.

Catabolite Repression

• Prevents the expression of genes needed for the catabolism of sugars (e.g., lactose, arabinose) in the presence of glucose.
• Mediated by cAMP and cAMP receptor protein (CRP).

Attenuation

• Depends on the availability of tryptophan.
• High Trp: tRNATrp is high, leading to attenuation; transcription stops due to the formation of a hairpin structure.
• Low Trp: tRNATrp is low, allowing for transcription to proceed; ribosome halts at Trp codons, enabling segment 2 and 3 to pair.

Prokaryotes vs. Eukaryotes

• Prokaryotes: circular chromosomes, less complex, one chromosome, lack introns, have exons, lack histones, and have operons.
• Eukaryotes: linear chromosomes, more complex, 23 chromosomes, have introns/exons, have histones, and have positive/negative regulators.

Regulatory Processes in Eukaryotes

• Gene alteration: changes made in the gene sequence affecting gene expression.
• Transcriptional control: regulates gene expression during transcription.
• Posttranscriptional control: regulates gene expression after transcription, including RNA splicing, alternative splicing, and RNA editing.
• Translational control: regulates gene expression during translation.
• Posttranslational control: modifies newly translated proteins via enzymes to their active conformation.

SOS Response and RNA-mediated Regulation

• SOS response: when excessive DNA damage triggers an induction of genes involved in stopping DNA replication and repair.
• SOS response involves RecA proteins and LexA repressor.
• RNA-mediated regulation: regulates the expression of genes involved in producing ribosomes (r-proteins) via translational feedback.

Eukaryotic Gene Regulation and Hormonal Regulation

• Eukaryotic gene regulation requires chromatin remodeling, activators, coactivators, architectural regulators, and transcriptional factors.
• Hormonal regulation: hormones bind to receptors, affecting transcription via Hormone Response Elements (HREs).
• Type I hormone receptors: nuclear receptors (e.g., sex hormones, glucocorticoids) that bind to hormones and move to the nucleus to activate transcription.
• Type II hormone receptors: thyroid hormone receptors found in the nucleus, which activate transcription when bound to hormones.

Iron Responsive Element (IRE) and Iron Responsive Factor (IRF)

• IRF binds to IREs in mRNAs, regulating the synthesis of transferrin receptors and ferritin.
• IREs: hairpin structures found on the ends of mRNA (5' for ferritin and 3' for transferrin receptor).
• Iron deficiency: IRP binds to IRE, increasing transferrin synthesis and decreasing ferritin synthesis.
• Iron excess (hemochromatosis): IRP does not bind to IRE, decreasing transferrin synthesis and increasing ferritin synthesis.

Stem Cells and Gene Regulation

• Stem cells can differentiate into various tissues and replenish themselves, regulated by signals.
• Embryonic stem cells are pluripotent, meaning they can differentiate into many tissues.

Clinical Correlation: Drug Resistance and Interferon

• Methotrexate: inhibits dihydrofolate reductase, preventing the production of tetrahydrofolate, essential for DNA/RNA synthesis, killing cancer cells.
• Interferon: signaling proteins released in the presence of viruses, inhibiting gene expression through two mechanisms:
  • Activating protein kinases to prevent viral protein synthesis.
  • Activating ribonuclease to break down viral RNA, reducing the availability of ribosomes for protein synthesis.

Understand the process of gene evolution and the Central Dogma of molecular biology, including DNA replication, transcription, and translation. Discover how genetic information flows from DNA to proteins.