Molecular Biology, Gene Expression, and Biotechnology: Study Guide

Central Dogma and Genetic Code

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: RNA to DNA (e.g., retroviruses, catalyzed by reverse transcriptase)

  • RNA Replication: RNA to RNA (e.g., RNA viruses, catalyzed by RNA-dependent RNA polymerase)

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

Genetic Code

• The genetic code is redundant (multiple codons for one amino acid), unambiguous (each codon specifies only one amino acid), and universal (shared by most organisms).

• Transcription: Given a DNA template, the mRNA sequence is synthesized by pairing complementary bases (A-U, T-A, C-G, G-C).

• Translation: mRNA codons are read by the ribosome to assemble amino acids into a polypeptide chain.

• Example: DNA coding strand: 5'-ATG GGC TAA-3' → mRNA: 5'-AUG GGC UAA-3' → Amino acids: Met-Gly-Stop

Mutations

• Silent Mutation: Changes a codon but not the amino acid

• Missense Mutation: Changes one amino acid in the protein

• 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 depends on the mutation type and location within the gene

Transcription and Translation

Transcription in Bacteria

• Initiation: RNA polymerase binds to the promoter region, unwinds DNA, and begins RNA synthesis

• Termination: Occurs when RNA polymerase encounters a terminator sequence, releasing the RNA transcript

RNA Processing in Eukaryotes

• Splicing: Removal of introns and joining of exons by the spliceosome

• 5' Capping: Addition of a modified guanine nucleotide to the 5' end for stability and ribosome recognition

• Polyadenylation: Addition of a poly(A) tail to the 3' end for stability and export from the nucleus

tRNA Structure and Function

• Amino Acid Attachment Site: 3' end of tRNA where the amino acid is covalently attached

• Anticodon: Three-nucleotide sequence that pairs with the mRNA codon during translation

• Function: tRNAs deliver specific amino acids to the ribosome based on codon-anticodon pairing

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 at the third codon position

Translation Process

• Initiation: Ribosome assembles at the start codon (AUG) on mRNA

• Elongation: tRNAs bring amino acids, and the polypeptide chain grows

• Termination: Stop codon is reached, and the completed polypeptide is released

Comparison: Bacteria vs. Eukaryotes

• Transcription and Translation: Coupled in bacteria, separated by the nuclear envelope in eukaryotes

• RNA Processing: Extensive in eukaryotes (capping, splicing, polyadenylation), minimal in bacteria

Viruses

Are Viruses Alive?

• Arguments for: Contain genetic material, evolve, reproduce (inside host)

• Arguments against: Not cellular, lack metabolism, cannot reproduce independently

Viral Life Cycle

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

Viral Genome Replication

• Depends on genome type (DNA or RNA, single or double stranded)

• May require host or viral enzymes (e.g., DNA polymerase, RNA-dependent RNA polymerase, reverse transcriptase)

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: The lac repressor binds the operator to block transcription; inducer (allolactose) inactivates the repressor

• Glucose Regulation: (1) Catabolite repression via cAMP-CRP, (2) Inducer exclusion

• Mutations: Affect expression (e.g., nonfunctional repressor, operator mutations)

trp Operon

• Repressible operon; tryptophan acts as a corepressor

Comparison: lac vs. trp Operons

• lac: Inducible, usually off, turned on by inducer

• trp: Repressible, usually on, turned off by corepressor

Regulation of Gene Expression in Eukaryotes

Chromatin Remodeling

• Chromatin structure affects gene accessibility

• Remodeling complexes reposition nucleosomes; histone modifications (acetylation, methylation) regulate transcription

Transcription Initiation

• Requires transcription factors, mediator complex, and RNA polymerase II

Alternative Splicing

• Allows one gene to produce multiple protein isoforms by including/excluding different exons

RNA Interference (RNAi)

• Small RNAs (siRNA, miRNA) bind mRNA 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 regulation (chromatin, RNA processing, RNAi, 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 bacteria containing the plasmid

• Steps: (1) Cut DNA and plasmid, (2) Ligate, (3) Transform bacteria, (4) Select colonies

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, dNTPs, Taq polymerase, buffer

• Steps: (1) Denaturation, (2) Annealing, (3) Extension

Agarose Gel Electrophoresis

• Separates DNA fragments by size

• Used for DNA fingerprinting and analysis

Sanger Sequencing

• ddNTPs: Dideoxynucleotides terminate DNA synthesis, allowing sequence determination

Genomics

• Genome studies reveal gene functions, evolutionary relationships, and genetic variation

Genes, Development, and Stem Cells

Genetic Equivalence

• All cells in an organism contain the same DNA

• Evidence: Cloning experiments in plants and animals

Cell Differentiation

• Process by which cells become specialized in structure and function

• Regulated by gene expression patterns

Stem Cells

• Adult Stem Cells: Multipotent, limited differentiation potential

• Embryonic Stem Cells: Pluripotent, can become any cell type

• Induced Pluripotent Stem Cells (iPS): Adult cells reprogrammed to pluripotency

• Stem Cell Therapy: Potential to treat diseases by replacing damaged cells