Study Guide: Central Dogma, DNA Replication, Mutation, Transcription, RNA Processing, Translation, and Genetic Code

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Central Dogma of Molecular Biology

Overview of the Central Dogma

The central dogma of molecular biology describes the flow of genetic information within a biological system. It states that information passes from DNA to RNA to protein, encompassing the processes of DNA replication, transcription, and translation.

DNA Replication: The process by which DNA makes a copy of itself.
Transcription: The synthesis of RNA from a DNA template.
Translation: The synthesis of proteins from an RNA template.
Extensions: Reverse transcription (RNA to DNA), RNA replication (RNA to RNA), and non-coding RNAs.

Example: Retroviruses use reverse transcription to convert their RNA genome into DNA.

Features of a Gene Enabling Transcription and Translation

A gene contains specific sequences that allow for its expression:

Promoter: DNA sequence where RNA polymerase binds to initiate transcription.
Coding Region: Sequence that is transcribed and translated into protein.
Regulatory Elements: Enhancers, silencers, and operators that modulate gene expression.
Start and Stop Codons: Define the beginning and end of translation.

Differences in Central Dogma: Eukaryotes vs Prokaryotes

Eukaryotes: Transcription occurs in the nucleus; translation occurs in the cytoplasm. RNA processing (capping, splicing, polyadenylation) is required.
Prokaryotes: Transcription and translation are coupled in the cytoplasm; minimal RNA processing.

Transcription vs Translation

Transcription: Copying DNA sequence into RNA (same "language" of nucleotides).
Translation: Converting RNA sequence into protein (changing "language" from nucleotides to amino acids).

Protein Structure

Primary Structure: Linear sequence of amino acids.
Secondary Structure: Local folding (α-helix, β-sheet).
Tertiary Structure: Overall 3D shape.
Quaternary Structure: Association of multiple polypeptides.

DNA Replication

Models of DNA Replication

Three models were proposed:

Conservative: Parent DNA remains intact; new molecule is entirely new DNA.
Semiconservative: Each new DNA molecule contains one old and one new strand.
Dispersive: DNA strands are mixtures of old and new segments.

Meselson and Stahl Experiment: Used isotopic labeling to show DNA replication is semiconservative.

Requirements for DNA Polymerase

Template DNA: Provides the sequence to copy.
Primer: Short RNA sequence to start synthesis.
dNTPs: Deoxynucleotide triphosphates as substrates.

Directionality of DNA Replication

DNA polymerase synthesizes DNA in the 5' to 3' direction, meaning new nucleotides are added to the 3' end.

Chemical Reaction of DNA Polymerization

Formation of phosphodiester bonds between nucleotides.
Hydrolysis of pyrophosphate provides energy.

Enzymes in DNA Replication

Helicase: Unwinds DNA helix.
Primase: Synthesizes RNA primers.
Gyrase (Topoisomerase): Relieves supercoiling.
DNA Polymerase I: Removes RNA primers and fills gaps.
Flap Endonuclease: Removes RNA flaps during lagging strand synthesis.
DNA Ligase: Seals nicks in DNA backbone.

Leading and Lagging Strand Synthesis

Leading Strand: Synthesized continuously toward replication fork.
Lagging Strand: Synthesized discontinuously as Okazaki fragments away from fork.
Both strands are synthesized simultaneously from a replication bubble.

End Replication Problem and Telomerase

Linear chromosomes cannot be fully replicated at ends by DNA polymerase.
Telomerase: Adds repetitive sequences to chromosome ends, solving the problem.
Expressed in germ cells, stem cells, and some cancer cells; not in most somatic cells.
Cancer cells often reactivate telomerase to enable unlimited division.

Mutation and Cancer

Types of Mutations

Somatic Mutations: Occur in non-reproductive cells; not inherited.
Germline Mutations: Occur in gametes; can be inherited.
Point Mutations: Single nucleotide changes (e.g., transitions, transversions).
Structural Mutations: Large-scale changes (e.g., deletions, duplications, inversions).

Causes and Consequences of Mutations

Can arise from errors in replication, environmental factors, or spontaneous chemical changes.
Consequences range from neutral to deleterious or beneficial.

Categorizing Mutations

Type: Point, structural, insertion, deletion.
Target: Coding region, regulatory region.
Effect: Silent, missense, nonsense, frameshift.

Randomness of Mutations

Luria-Delbruck Experiment: Demonstrated mutations occur randomly, not in response to selective pressure.

Nirenberg Experiment: Helped elucidate the genetic code; also showed randomness in mutation with respect to fitness.

Transitions vs Transversions

Transitions: Purine to purine (A ↔ G) or pyrimidine to pyrimidine (C ↔ T).
Transversions: Purine to pyrimidine or vice versa.
Transitions are more common due to molecular mechanisms (e.g., tautomeric shifts).

Proto-oncogenes vs Tumor Suppressor Genes

Proto-oncogenes: Promote cell growth; mutations can lead to cancer (oncogenes).
Tumor Suppressor Genes: Inhibit cell growth; loss-of-function mutations can lead to cancer.
Tumor suppressor gene mutations are more likely to be inherited.

Two-Hit Model of Cancer Development

Both alleles of a tumor suppressor gene must be inactivated for cancer to develop.
First hit may be inherited; second hit occurs somatically.

Transcription: Making RNA from DNA

Template and Coding Strands

Template (Anti-sense) Strand: Used by RNA polymerase to synthesize RNA.
Coding (Sense) Strand: Sequence matches RNA (except T replaced by U).
Knowing one strand allows prediction of the other and the RNA sequence.

RNA vs DNA Polymerases

RNA Polymerase: Synthesizes RNA; does not require a primer.
DNA Polymerase: Synthesizes DNA; requires a primer.
Both catalyze phosphodiester bond formation.

Transcription Initiation and Termination in Prokaryotes

Initiation: Sigma factor binds promoter, recruits RNA polymerase.
Termination: Rho factor binds Rut site, causes release of RNA.

Transcription Initiation and Termination in Eukaryotes

Initiation: TATA box recognized by TFIID, TFIIB, TFIIH, and other transcription factors; Mediator complex assembles.
Termination: PolyA-site recognized; terminating endonucleases cleave RNA.

RNA Processing in Eukaryotes

Three Features of RNA Processing

5' Cap: Added early during transcription; protects RNA and aids translation.
Splicing: Removes introns; performed by spliceosome.
3' Poly-A Tail: Added after transcription; stabilizes RNA.

Introns, Exons, and Spliceosome Function

Introns: Non-coding regions removed from pre-mRNA.
Exons: Coding regions retained in mature mRNA.
Spliceosome: Complex that catalyzes splicing.

Alternative Splicing

Allows production of multiple proteins from one gene.
Important for functional diversity.

Polyadenylation and Transcription Termination

Poly-A tail added at 3' end after cleavage at polyA-site.
Linked to termination of transcription.

Translation: Making Proteins from RNA

Directionality and Chemical Terminology

DNA/RNA: Synthesized 5' to 3'.
Polypeptide: Synthesized amino (N) to carboxyl (C) terminus.
tRNA: Matches codons to amino acids during translation.

Translation Initiation: Prokaryotes vs Eukaryotes

Prokaryotes: Shine-Dalgarno sequence aligns ribosome.
Eukaryotes: 5' Cap and Kozak sequence guide ribosome to start codon.

Ribosome Sites and tRNA Function

A-site: Accepts incoming tRNA.
P-site: Holds tRNA with growing polypeptide.
E-site: Exit site for tRNA.
tRNA structure enables accurate translation of mRNA codons.

Translation Termination

Release Factor: Recognizes stop codon, releases polypeptide.

Protein Sequence and Function

Sequence of amino acids determines protein folding and function.

The Genetic Code

Reading Frame

Translation reads mRNA in sets of three nucleotides (codons).
Reading frame determined by translation initiation site.

Degeneracy of the Genetic Code

Multiple codons can code for the same amino acid.
Three-letter code hypothesized due to 20 amino acids and 4 nucleotides.

possible codons

Determination of the Genetic Code

Khorana and Nirenberg used synthetic RNAs to determine which codons specify which amino acids.
Data interpretation involves matching codon sequences to amino acid incorporation.

Table: Example Codon Assignments (inferred from Khorana/Nirenberg data)

Codon	Amino Acid
UUU	Phenylalanine
AUG	Methionine (Start)
UAA	Stop
GCU	Alanine
GGG	Glycine
Additional info: ...	Other codons determined experimentally

Example: The codon UUU codes for phenylalanine, as shown by Nirenberg's experiments.

Additional info: Academic context was added to clarify mechanisms, enzyme functions, and experimental details for completeness.