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

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 → RNA: Transcription (enzyme: RNA polymerase)

• RNA → Protein: Translation (enzyme: ribosome)

Exceptions to the Central Dogma:

• Reverse Transcription: RNA → DNA (enzyme: reverse transcriptase; e.g., retroviruses)

• RNA Replication: RNA → RNA (enzyme: RNA-dependent RNA polymerase; e.g., some viruses)

• Non-coding RNAs: Some RNAs (e.g., rRNA, tRNA, miRNA) are not translated into proteins but have functional roles.

Flow Chart:

• DNA --(transcription)--> RNA --(translation)--> Protein

• Exceptions: RNA --(reverse transcription)--> DNA; RNA --(replication)--> RNA; RNA (functional, not translated)

Transcription and Translation

• Transcription: Synthesis of mRNA from a DNA template.

• Translation: Synthesis of a polypeptide (protein) from an mRNA template.

Determining Sequences:

• Given a DNA coding or template strand, the mRNA sequence can be determined by base pairing rules (A-U, T-A, C-G, G-C).

• Given a DNA or mRNA sequence, the amino acid sequence is determined using the genetic code table.

The Genetic Code

• Redundant: Multiple codons can code for the same amino acid.

• Unambiguous: Each codon specifies only one amino acid.

• Universal: The code is nearly universal across all organisms.

Types of Mutations

• Silent Mutation: No change in amino acid sequence.

• Missense Mutation: Changes one amino acid to another.

• Nonsense Mutation: Changes a codon to a stop codon, truncating the protein.

• Frameshift Mutation: Insertion or deletion shifts the reading frame, altering downstream amino acids.

Predicting Consequences: The effect of a mutation depends on its type and location within the gene.

Transcription, RNA Processing, and Translation

Transcription in Bacteria

• Initiation: RNA polymerase binds to the promoter region.

• Termination: RNA polymerase stops transcription at a terminator sequence.

RNA Processing in Eukaryotes

• Splicing: Removal of introns and joining of exons.

• 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; binds specific amino acid.

• Anticodon: Triplet sequence that base-pairs with mRNA codon.

Function: tRNAs deliver amino acids to the ribosome during translation.

Aminoacyl-tRNA Synthetases (ARSs)

• Enzymes that attach amino acids to their corresponding tRNAs.

• Fewer ARSs and tRNAs than codons due to wobble pairing.

Translation Steps

• Initiation: Assembly of ribosome, mRNA, and initiator tRNA.

• Elongation: Addition of amino acids to the growing chain.

• Termination: Release of the completed polypeptide when a stop codon is reached.

Comparison: Bacteria vs. Eukaryotes

• Transcription: Coupled with translation in bacteria; separated in eukaryotes.

• RNA Processing: Extensive in eukaryotes; minimal in bacteria.

• Translation: Similar mechanism, but different ribosome structures.

Viruses

Are Viruses Alive?

• Arguments for: Reproduce, evolve, contain genetic material.

• Arguments against: Not cellular, lack metabolism, require host for replication.

Viral Life Cycle

• Entry: Virus 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 with it; can later enter lytic cycle.

Viral Genome Replication

• Depends on genome type (DNA or RNA) and host/viral enzymes required.

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

Transcriptional Regulation in Bacteria

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

lac Operon

• Negative Control: lac repressor binds operator, blocking transcription in absence of inducer (lactose).

• Inducer Present: Repressor is inactivated, allowing transcription.

• Mutations: Can affect operon expression (e.g., nonfunctional repressor, operator mutations).

• Glucose Regulation: Catabolite repression (cAMP-CAP complex) and inducer exclusion.

trp Operon

• Repressible operon; tryptophan acts as a corepressor.

Comparison: lac vs. trp Operons

• lac: Inducible, usually off, turned on by substrate (lactose).

• trp: Repressible, usually on, turned off by product (tryptophan).

Gene Expression in Eukaryotes

Chromatin Remodeling

• Modification of chromatin structure (e.g., histone acetylation) affects transcriptional accessibility.

Transcription Initiation

• Requires transcription factors and RNA polymerase binding to promoter.

Alternative Splicing

• Allows one gene to produce multiple protein isoforms by varying exon inclusion.

RNA Interference (RNAi)

• Small RNAs (e.g., siRNA, miRNA) bind mRNAs and block translation or promote degradation.

Protein Degradation

• Ubiquitin: Tags proteins for degradation.

• Proteasome: Degrades ubiquitinated proteins.

Comparison: Bacteria vs. Eukaryotes

• Eukaryotes have more complex, multi-level regulation (chromatin, RNA processing, etc.).

Biotechnology and Genomics

DNA Cloning

• Plasmids: Circular DNA vectors for gene insertion.

• Restriction Enzymes: Cut DNA at specific sequences.

• DNA Ligase: Joins DNA fragments.

• Antibiotic Resistance Gene: Selects for cells containing plasmid.

Steps of DNA Cloning

1. Cut DNA and plasmid with restriction enzymes.

2. Ligate DNA fragment into plasmid.

3. Transform plasmid into bacteria.

4. Select for transformants using antibiotic resistance.

CRISPR-Cas System

• Bacterial defense mechanism against viruses.

• CRISPR-Cas9 can be used for gene editing or inactivation.

• Components: Cas9 protein, guide RNA, target DNA.

PCR (Polymerase Chain Reaction)

• Components: Template DNA, primers, DNA polymerase, dNTPs, buffer.

• Steps:

  1. Denaturation: DNA strands separate.

  2. Annealing: Primers bind to target sequence.

  3. Extension: DNA polymerase synthesizes new DNA.

Agarose Gel Electrophoresis

• Separates DNA fragments by size; smaller fragments move farther.

• Used in DNA fingerprinting and analysis.

Sanger Sequencing

• ddNTPs: Dideoxynucleotides terminate DNA synthesis, allowing sequence determination.

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.

Cell Differentiation

• Process by which cells become specialized in structure and function.

Stem Cells

Type	Source	Potency	Applications
Adult Stem Cells	Adult tissues	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.