Gene Expression, Regulation, Viruses, Biotechnology, and Development: Study Notes

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Gene Expression and the Central Dogma

The Central Dogma of Molecular Biology

The central dogma describes the flow of genetic information within a biological system. It outlines how DNA is transcribed into RNA, which is then translated into protein.

DNA --(transcription via RNA polymerase)--> RNA --(translation via ribosome)--> Protein
Each arrow represents a process catalyzed by a specific enzyme:
- Transcription: DNA to RNA, catalyzed by RNA polymerase
- Translation: RNA to protein, catalyzed by the ribosome

Exceptions to the Central Dogma:

Reverse Transcription: Some viruses (retroviruses) use reverse transcriptase to synthesize DNA from RNA.
RNA Replication: Some RNA viruses replicate their RNA genomes directly using RNA-dependent RNA polymerase.
Non-coding RNAs: Some RNAs (e.g., rRNA, tRNA, miRNA) are not translated into proteins but have functional roles.

Transcription and Translation: From DNA to Protein

Transcription: The process of synthesizing mRNA from a DNA template.
Translation: The process of synthesizing a polypeptide (protein) from an mRNA template.
Determining mRNA Sequence: Given a DNA template, replace A with U, T with A, C with G, and G with C to get the mRNA sequence.
Determining Amino Acid Sequence: Use the genetic code to translate mRNA codons into amino acids.

The Genetic Code

Redundant: Multiple codons can code for the same amino acid.
Unambiguous: Each codon specifies only one amino acid.
Universal: The genetic code is nearly universal across all organisms.

Types of Mutations

Silent Mutation: Changes a codon but does not alter the amino acid sequence.
Missense Mutation: Changes a codon, resulting in a different amino acid.
Nonsense Mutation: Changes a codon to a stop codon, truncating the protein.
Frameshift Mutation: Insertion or deletion of nucleotides that shifts the reading frame.

Example: If the DNA sequence AAA (Lys) is mutated to TAA (stop), this is a nonsense mutation.

Transcription and Translation in Prokaryotes and Eukaryotes

Transcription in Bacteria

Initiation: RNA polymerase binds to the promoter region.
Termination: Occurs via rho-dependent or rho-independent mechanisms.

RNA Processing in Eukaryotes

Splicing: Removal of introns and joining of exons by the spliceosome.
Capping: Addition of a 5' methylguanosine cap for stability and translation initiation.
Polyadenylation: Addition of a poly(A) tail at the 3' end for stability and export.

tRNA Structure and Function

Amino Acid Attachment Site: 3' end of tRNA where the amino acid is covalently attached.
Anticodon: A three-nucleotide sequence that pairs with the mRNA codon.

Aminoacyl-tRNA Synthetases (ARSs)

Enzymes that attach the correct amino acid to its corresponding tRNA.
There are fewer ARSs and tRNAs than codons due to wobble pairing.

Translation: Initiation, Elongation, Termination

Initiation: Assembly of the ribosome on the mRNA with initiator tRNA.
Elongation: Sequential addition of amino acids.
Termination: Release of the polypeptide when a stop codon is reached.

Comparison: Bacteria vs. Eukaryotes

Bacteria: Transcription and translation are coupled; no RNA processing.
Eukaryotes: Transcription in nucleus, translation in cytoplasm; extensive RNA processing.

Viruses: Structure, Life Cycle, and Classification

Are Viruses Alive?

Arguments for: Contain genetic material, evolve, reproduce (inside host).
Arguments against: Not cellular, no metabolism, require host for replication.

Viral Life Cycle

Entry: Virus attaches and enters host cell.
Gene Expression: Viral genes are transcribed and translated.
Assembly: New viral particles are assembled.
Exit: Viruses leave the host cell (lysis or budding).

Lytic vs. Lysogenic/Latency Cycles

Lytic Cycle: Virus replicates and lyses host cell.
Lysogeny/Latency: Viral genome integrates into host DNA and is replicated passively.

Viral Genome Replication

Depends on genome type (DNA or RNA) and may require host or viral enzymes.

Antiviral Drugs and Vaccines

Antiviral Drugs: Inhibit viral replication (e.g., reverse transcriptase inhibitors).
Vaccines: Stimulate immune response to prevent infection.

Regulation of Gene Expression in Bacteria

Transcriptional Regulation

Bacteria primarily regulate gene expression at the transcriptional level for efficiency.

lac Operon: Negative Control

In absence of inducer (lactose): Repressor binds operator, blocking transcription.
In presence of inducer: Inducer binds repressor, allowing transcription.

Mutations in the lac Operon

Mutations can affect expression (e.g., nonfunctional repressor leads to constitutive expression).

Glucose Regulation of the lac Operon

Catabolite repression via cAMP-CAP complex; glucose inhibits cAMP, reducing transcription.
Inducer exclusion: Glucose transport inhibits lactose import.

Comparison: lac vs. trp Operon

lac Operon: Inducible; turned on by presence of lactose.
trp Operon: Repressible; turned off by presence of tryptophan.

Regulation of Gene Expression in Eukaryotes

Chromatin Remodeling

Modification of histones (acetylation, methylation) alters DNA accessibility and transcription.

Transcription Initiation in Eukaryotes

Requires assembly of transcription factors and RNA polymerase at the promoter.

Alternative Splicing

Allows a single gene to produce multiple protein isoforms by varying exon inclusion.

RNA Interference (RNAi)

Small RNAs (siRNA, miRNA) bind mRNA and block translation or promote degradation.

Protein Degradation: Ubiquitin and Proteasome

Proteins tagged with ubiquitin are degraded by the proteasome.

Comparison: Bacterial vs. Eukaryotic Gene Regulation

Bacteria: Primarily transcriptional regulation.
Eukaryotes: Multiple levels—chromatin, transcription, RNA processing, translation, and protein degradation.

Biotechnology: DNA Cloning, PCR, and Genome Analysis

DNA Cloning Components

Plasmids: Circular DNA vectors for gene insertion and propagation.
Restriction Enzymes: Cut DNA at specific sequences.
DNA Ligase: Joins DNA fragments together.
Antibiotic Resistance Gene: Selects for bacteria containing the plasmid.

Steps of DNA Cloning

Cut DNA and plasmid with restriction enzymes.
Ligate DNA fragment into plasmid.
Transform bacteria with recombinant plasmid.
Select for transformants using antibiotic resistance.

CRISPR-Cas System

Bacterial defense mechanism against viruses; uses guide RNA and Cas9 protein to target and cut foreign DNA.
CRISPR-Cas9 can be engineered for gene editing in other organisms.

PCR (Polymerase Chain Reaction)

Components: Template DNA, primers, dNTPs, Taq polymerase, buffer.
Steps:
- Denaturation: to separate DNA strands.
- Annealing: for primers to bind.
- Extension: for DNA synthesis.

Agarose Gel Electrophoresis

Separates DNA fragments by size; smaller fragments move further.
Used for DNA fingerprinting and analysis.

Sanger Sequencing and ddNTPs

ddNTPs (dideoxynucleotides): Chain-terminating nucleotides used to determine DNA sequence.

Genomics

Study of whole genomes; reveals gene content, organization, and evolutionary relationships.

Development and Stem Cells

Genetic Equivalence

All cells in an organism contain the same DNA; differences arise from gene expression.
Evidence: Cloning experiments in plants and animals (e.g., Dolly the sheep).

Cell Differentiation

Process by which cells become specialized in structure and function.

Stem Cells

Type	Source	Potency	Applications
Adult Stem Cells	Tissues (e.g., bone marrow)	Multipotent	Tissue repair
Embryonic Stem Cells	Blastocyst	Pluripotent	Regenerative medicine
iPS Cells	Reprogrammed adult cells	Pluripotent	Personalized therapy

Stem cell therapy uses stem cells to replace or repair damaged tissues.