Chapter 6 - Gene Expression at the Molecular Level

Gene Expression at the Molecular Level I

• Gene expression is the process of converting genetic information into a functional product
• It is studied at both the molecular and trait levels
• Mutations are heritable changes in genetic material affecting gene function by altering the gene sequence
• Research on mutations has shown the relationship between normal genes and functional proteins, and abnormal genes and non-functional proteins.
• Inborn Errors of Metabolism are inherited diseases where abnormal levels of homogentisic acid accumulate, demonstrating a link between mutant genes, defective enzymes, and metabolic diseases.

Archibald Garrod

• Studied patients with metabolic defects, such as alkaptonuria
• In 1908, proposed a link between a mutant gene, missing/defective enzyme and metabolic disease, calling it an "inborn error of metabolism"

Phenylalanine Metabolism

• Mutations can cause phenylketonuria or tyrosinosis or alkaptonuria
• Mutations in genes related to these pathways cause metabolic disorders

Beadle and Tatum

• Discovered Garrod's work in the early 1940s
• Studied Neurospora crassa (common bread mold)
• Determined minimum growth requirements
• Focused on amino acid synthesis
• Hypothesized that genes encode enzymes, and mutations can cause defects in enzymes needed for amino acid synthesis.
• Proposed that each step in a biochemical pathway is catalyzed by a different enzyme, controlled by a different gene.
• Collected mutant strains needing arginine supplementation for growth
• Explored precursor molecules' effects on mutant strain growth
• Identified three genes and enzymes for arginine synthesis
• Found mutant strains grouped according to defective enzymes. Demonstrated single gene controlling synthesis of single enzyme

The Central Dogma of Gene Expression

• The fundamental principle of gene expression
• Shows the flow of genetic information from DNA to RNA to protein
• Includes processes like replication, transcription, and translation

Prokaryotes vs. Eukaryotes

• Prokaryotes have simpler gene expression processes. Transcription in the cytoplasm, no modifications occur. Translation takes place immediately.
• Eukaryotes are more complex, with transcription in the nucleus and subsequent RNA modifications (splicing etc), and separate translation in the cytoplasm.

Genes

• Organized units of DNA sequences transcribed into RNA
• Protein-coding genes produce mRNA specifying amino acid sequences, to eventually make proteins
• Non-coding RNA genes (e.g., tRNA, rRNA) produce functional RNA molecules

Gene Organization

• DNA segments organized into promoter, transcribed region, and terminator (signals for transcription start and end)
• Regulatory sequences influence transcription rate
• The transcribed region carries the amino acid sequence coding information

Three Stages of Transcription

• Initiation - RNA polymerase binds to promoter using sigma factor, forming an open complex
• Elongation - RNA polymerase moves along template strand, creating RNA in 5' to 3' direction, complementary to template
• Termination - RNA polymerase reaches the terminator sequence, and the RNA transcript and RNA polymerase dissociate from DNA template

A Closer Look at Elongation

• RNA synthesis is 5' to 3'
• Uracil (U) in RNA replaces Thymine (T) of DNA.
• Template strand of DNA is used as a template and read in the 3' to 5' direction
• Coding strand has same sequence as mRNA, except T instead of U

Transcription along the Chromosome

• DNA strand used as template can differ for adjacent genes.
• Determined by promoter sequence location

Eukaryotic Transcription

• Eukaryotes contain three RNA polymerases instead of one, and need several initiation factors
• Basic features are identical, but each step involves greater complexity compared to prokaryotes.

Eukaryotic RNA Processing

• Pre-mRNA undergoes capping, splicing, and polyadenylation
• Capping involves adding a 5' cap (7-methylguanosine)
• Splicing removes non-coding introns, joining exons
• Polyadenylation involves adding a poly-A tail at the 3' end

RNA Processing: Capping

• Covalent attachment of 7-methylguanosine to the 5' end of the mRNA transcript (5' cap)
• Occurs while RNA polymerase synthesizes the pre-mRNA
• Recognized by cap-binding proteins
• Needed for mRNA nucleus exit
• Protects mRNA and binds to ribosome for translation

RNA Processing: Tailing

• Adds poly-A tail (100-200 adenines) to the 3' end of the mRNA
• Helps with export from the nucleus and enhances mRNA stability
• Allows mRNA to persist longer in the cytoplasm

RNA Processing: Splicing

• Removes non-coding introns and joins exons
• Catalyzed by the spliceosome (snRNPs)
• snRNPs are made of snRNA & protein
• Alternative splicing allows different gene products through variation on how exons are joined

The Genetic Code

• Specifies the relationship between mRNA nucleotide sequence and corresponding polypeptide amino acid sequence
• Codons are three-nucleotide sequences specifying amino acids or signaling start/stop
• Examples: CCC = proline, GGC = glycine
• tRNAs bind to mRNA codons using anticodons

The Genetic Code: Degeneracy

• Multiple codons may specify the same amino acid
• Using 3 bases, 64 different codons are possible (4^3). But there are only 20 different amino acids

Bacterial mRNA Organization

• Contains ribosomal-binding site, start codon, coding sequence, and stop codon
• Specified to code for specific amino acid sequence

Reading Frame

• Start codon defines the reading frame
• Adjacent codons are read as triplets in a 5' -> 3' direction
• Insertions/deletions (not multiple of 3) lead to altered amino acid sequences

The Translation Machinery

• mRNA carries the genetic code for amino acid sequence
• Ribosomes are the sites of protein synthesis, composed of proteins and rRNA molecules, and have A, P, and E sites
• tRNAs carry amino acids, with anticodons that match mRNA codons

tRNAs

• Diverse tRNAs encoded by different genes, sharing common features, and a cloverleaf structure
• Have an anticodon to recognize their mRNA codon
• Acceptor stem binds amino acid

Aminoacyl-tRNA Synthetases

• Enzymes attach amino acids to tRNA molecules.
• There is one amino acid-specific synthetase for each of the 20 amino acids
• Two-step reaction to create charged (aminoacyl-tRNA)

Ribosomes

• Macromolecular structures where translation takes place.
• Composed of proteins and rRNAs forming large/small subunits
• Eukaryotic ribosomes combine 40S and 60S to form 80s ribosomes, prokaryotic are 30S and 50S forming 70S

Ribosome Structure

• rRNA determines overall ribosome shape
• tRNAs bind to A, P, and E sites for polypeptide elongation

Three Stages of Translation

• Initiation - mRNA, tRNA, and ribosomal subunits combine
• Elongation - Ribosome moves along mRNA, forming polypeptide chain
• Termination - Stop codon signals the end of translation and release of the polypeptide chain

Translation Initiation in Bacteria

• mRNA binds to the small ribosomal subunit
• Initiator tRNA binds to the start codon (AUG)
• Large ribosomal subunit binds to form the complete initiation complex

Eukaryotic Initiation

• mRNAs lack ribosomal binding sites
• Cap-binding proteins and initiation factors required
• Start codon is more variable and often the first AUG codon

Elongation -1

• Aminoacyl-tRNA binds to A site on ribosome
• Recognized by codon/anticodon matching
• Peptidyl tRNA is in P site

Elongation -2

• Peptide bond forms between amino acids at A and P sites
• Catalyzed by rRNA (ribozyme activity)
• Polypeptide chain moves from P site to A site

Elongation -3

• Ribosome shifts along the mRNA by one codon
• Uncharged tRNA moves from P to E site exiting
• New aminoacyl-tRNA enters A site

Termination

• Translation stops when a stop codon is reached in the A site
• Release factors bind and complete polypeptide release, leading to ribosome sub-unit dissociation