Transcription and Translation Notes

Genes and Proteins

• Genes contain information coded by nucleotide sequences (DNA bases).
• Inherited DNA dictates traits by directing protein synthesis.
• Proteins link genotype (genetic makeup) and phenotype (observable traits).
• Gene expression: DNA controls protein synthesis through transcription and translation.

Historical Perspective

• 1902: Archibald Garrod suggested genes dictate phenotype via enzymes catalyzing chemical reactions.
• Symptoms of disease reflect inability to synthesize certain enzymes.
• Cells synthesize and degrade molecules in metabolic pathways.
• 1940s: Beadle and Tatum experiment with bread mold to prove the gene-enzyme relationship.
• Used X-rays as a mutagen.

Beadle and Tatum Experiment

• Exposed bread mold to X-rays, generating mutants unable to survive on minimal growth media.
• Identified three classes of mutants deficient in arginine synthesis.
• Each mutant lacked a different enzyme in the arginine synthesis pathway.
• Developed the "one gene, one enzyme" hypothesis.
• Wild type organism grows on minimal media with or without ornithine, citrulline, or arginine.

Classes of Mutants

• Class I: Blocked in the first enzyme; grow only with ornithine, citrulline, or arginine.
• Class II: Mutation in the second enzyme; grow only with citrulline or arginine.
• Class III: Mutation in the third enzyme; grow only with arginine.

Evolution of the Hypothesis

• Revised to "one gene, one protein" as not all proteins are enzymes.
• Many proteins consist of multiple polypeptides.
• Current hypothesis: "one gene, one polypeptide".
• Gene products commonly referred to as proteins, acknowledging that multiple polypeptides may be needed for an active protein.

From Gene to Protein

• RNA is the intermediary molecule between DNA and protein.
• Transcription: Synthesis of messenger RNA (mRNA) from DNA.
• Translation: Synthesis of a polypeptide by ribosomes, using mRNA information.

Prokaryotes vs. Eukaryotes

• Prokaryotes: Transcription and translation occur in the cytoplasm.
• Translation of mRNA can begin before transcription finishes.
• Eukaryotes: Transcription in the nucleus; mRNA must exit to cytoplasm for translation.
• Transcription and translation are separated.

Central Dogma of Biology

• Linkage between DNA, RNA, and protein.
• Cells are governed by a cellular chain of command.

Genetic Code

• 20 amino acids, but only four nucleotide bases in DNA.
• Triplet code: Non-overlapping three-nucleotide words (codons) code for each amino acid.
• Codons are transcribed to complementary mRNA triplets.
• Ribosomes add amino acids to a polypeptide chain based on mRNA codons.

Triplet Codons

• 64 potential triplet codons.
• 61 code for amino acids, 3 are stop signals.
• Redundant: More than one codon may specify a particular amino acid.
• Unambiguous: No codon specifies more than one amino acid.
• Codons must be read in the correct reading frame for the correct polypeptide to be made.

Features of the Genetic Code

• Some amino acids have multiple codons (e.g., leucine has six).
• Methionine has only one codon.
• Third base wobble: Changes in the third base of a codon may not affect the amino acid specified.

Universality of the Genetic Code

• Shared by simplest bacteria to most complex animals.
• Genes can be transferred between species and still be transcribed and translated.

Transcription

• Copying DNA into RNA, catalyzed by RNA polymerase.
• RNA polymerase separates DNA strands and copies one strand into RNA nucleotides.
• RNA is complementary to the DNA template strand.
• RNA synthesis follows base pairing rules, except uracil (U) substitutes for thymine (T) in RNA.
• RNA polymerase doesn't need a primer.
• RNA polymerase binds to a DNA sequence called a promoter.

Transcription Process

• RNA polymerase binds to the promoter, separates DNA strands, and starts copying one strand into mRNA.
• It moves along the DNA, making the RNA transcript.
• Reaches a termination sequence, completing the RNA transcript.

RNA Processing in Eukaryotes

• mRNA is modified before leaving the nucleus.
• Both ends of the RNA transcript are altered and introns are removed (RNA splicing).

Altering mRNA Ends

• The five prime end receives a nucleotide cap.
• The three prime end gets a poly-A tail (50-250 adenine nucleotides).

Reasons for mRNA Modification

• Help export mRNA across the nuclear membrane.
• Protect mRNA from degradation by enzymes.
• Help ribosomes attach to the five prime end of the mRNA.

RNA Splicing

• Eukaryotic genes have non-coding introns and coding exons.
• RNA splicing removes introns and rejoins exons.
• Creates an mRNA with a continuous coding sequence.

Importance of Introns

• Some introns regulate gene expression.
• Alternative RNA splicing: Genes encode more than one polypeptide depending on which segments are treated as exons.
• Increases the number of different proteins an organism can produce.

Exons and Protein Domains

• Proteins have a modular architecture.
• Exons code for different functional domains in a protein.
• Exon shuffling can lead to the evolution of new proteins with different functions.

Protein Synthesis

• Making of proteins, essential for life because proteins perform transport, structural, enzymatic, protective, and other functions.
• DNA has genes that code for proteins to make pigments.
• Two major steps: transcription and translation.

Steps of Protein Synthesis

• Transcription: DNA is transcribed into mRNA in the nucleus by RNA polymerase.
• RNA polymerase connects complementary RNA bases to the DNA.
• mRNA is single-stranded and undergoes editing to be functional.
• mRNA exits the nucleus into the cytoplasm and attaches to a ribosome.

Translation

• Ribosomes, made of ribosomal RNA (rRNA), facilitate translation.
• Transfer RNA (tRNA) molecules carry amino acids (monomers of proteins).
• mRNA directs which tRNAs come in and therefore which amino acids are transferred.
• tRNAs look for complementary bases on the mRNA, reading in triplets called codons.

Codons and Anticodons

• Each tRNA contains a complementary anticodon to the mRNA codon.
• For example, if the mRNA codon is AUG, the tRNA anticodon is UAC.
• Specific tRNAs carry specific amino acids.
• Codon charts help determine which amino acid each mRNA codon will code for.
• AUG is a start codon and codes for methionine.

Codon Redundancy

• More possible codon combinations than types of amino acids.
• More than one codon can code for the same amino acid (e.g., leucine).

Translation Process

• tRNA brings in amino acids based on the mRNA sequence.
• Amino acids are held together by peptide bonds.
• Stop codons indicate the end of protein building.
• DNA directs the entire protein building process as mRNA is complementary to DNA.
• mRNA, rRNA, and tRNA are necessary for the protein building to occur.

Components of Translation

• mRNA is translated into protein by adding amino acids.
• Transfer RNA (tRNA) transfers amino acids to the growing polypeptide in the ribosome.
• Ribosomes have a large and small subunit; mRNA runs between the subunits.

tRNA Structure

• Small RNA molecule with a 2D and 3D structure.
• Anticodon recognizes a complementary three-base codon on the mRNA.
• Three-base codon codes for the amino acid at the three prime end, which it attaches to.

Ribosomes

• Couple tRNA anticodons with mRNA codons to make proteins.
• Made up of ribosomal RNA (rRNA) and over 30 different proteins.
• Small subunit has one rRNA molecule, and the large subunit has three rRNA molecules.
• Bacterial and eukaryotic ribosomes are similar but have significant differences.

Ribosome Binding Sites

• Three binding sites for tRNA: P (polypeptide growth), A (next amino acid), and E (exit).
• tRNAs match the codon of the mRNA, and used tRNAs leave.

Polypeptide Formation

• Polypeptide chain starts to coil and fold spontaneously to make a protein with a specific 3D shape.
• The gene from the DNA determines the primary structure, which determines the shape of the protein.

Multiple Ribosomes

• mRNA can be translated by multiple ribosomes simultaneously (polyribosome).
• Allows the cell to make many copies of a polypeptide quickly.

Prokaryotic Translation

• Transcription and translation happen at the same time.
• Ribosome can attach to mRNA while it's being transcribed.
• Eukaryotic translation is slowed down by physical separation and RNA processing by the nucleus.

Mutations

• Changes in the sequence that codes for proteins.
• Point mutations: Change in one base pair of a gene.
• Can be nucleotide pair substitutions, insertions, or deletions.
• Changes to a single nucleotide in a DNA template can produce abnormal proteins.
• Adverse effects on the phenotype of the organism can occur.

Types of Mutations

• Silent change: Change in base has no change on amino acid produced.
• Missense: Which changes the amino acid.
• Nonsense mutation: Codes for a stop codon.
• Insertions and deletions: The addition or removal of bases can have more significant effects than substitutions.
• Frameshift mutation: Adding an extra base causes a mutation with a stop codon being made.

Causes of Mutations

• During DNA replication.
• Via recombination.
• During incorrect DNA repair.
• Exposure to mutagens (physical or chemical agents that cause mutations).

DNA Repair

• DNA in cells is damaged thousands of times per day.
• Errors can affect DNA which can cause cancer.
• Damage comes in different forms like damaged nucleotides.

Repair Pathways

• Enzymes intersperse the damaged and undamaged strands.
• Homologous recombination: Enzymes use undamaged DNA as a template and get damaged and undamaged strands to exchange sequences of nucleotides.
• Nonhomologous end joining: Fuses broken ends but isn't accurate; can cause mixed-up genes.

Evolving Definition of a Gene

• Discrete unit of inheritance.
• Region of specific nucleotide sequence in a chromosome.
• DNA sequence that codes for a specific polypeptide chain.
• Region of DNA that can be expressed to produce a final functional product (polypeptide or RNA molecule).

RNA Modification After Transcription in Eukaryotic Cells

• PolyA tail added; five prime cap added; splicing to remove introns.
• Bacteria don't have post transcriptional steps because there is no nucleus.
• Transcription and translation can occur simultaneously, speeding up the process.