Announcements 1 Homework problem set 5 due this
Announcements 1. Homework - problem set 5 - due this Friday 2. Reading Ch. 14: Skim btm 391 -top 397. 3. Skip rest of 397 - 403.
Review of Last Lecture I. t. RNA and the genetic code II. Transcription - prokaryotes III. Transcription - eukaryotes
Outline of Lecture 25 I. RNA processing in eukaryotes II. Translation of m. RNA into protein - t. RNA and ribosomes III. Three steps of translation IV. First evidence that proteins are important to heredity V. One gene- one enzyme hypothesis
I. RNA Processing in Eukaryotes STABILITY
STABILITY
Introns and Exons
Eukaryotic vs. Prokaryotic Transcription • In eukaryotes, transcription and translation occur in separate compartments. • In bacteria, m. RNA is polycistronic; in eukaryotes, m. RNA is usually monocistronic. – Polycistronic: one m. RNA codes for more than one polypeptide – moncistronic: one m. RNA codes for only one polypeptide • 3 RNA polymerases in euk. , 1 in prok. • Binding of Basal Transcription Factors required for euk. RNA Pol II binding. • “Processing” of m. RNA in eukaryotes, no processing in prokaryotes
II. Structure: Unusual Bases Found in t. RNA
Function of Unusual Bases • Created post-transcriptionally. • Purpose is sometimes to allow for promiscuous basepairing: Inosine in the 1 st “wobble” position of anticodon can bind to 3 rd U, C or A in codon. • This means that fewer different t. RNAs are required. • Others play a structural role.
t. RNA Structure Aminoacyl t. RNA synthetase
Aminoacyl t. RNA Synthetases • Enzymes which bond specific amino acids to their cognate t. RNAs. • There are 20 different synthetases, one for each amino acid. • Covalent linkage through an ester bond (amino acid activation) requires ATP. • t. RNA linked to amino acid is charged.
Ribosome Structure S = Svedberg, a measure of sedimentation in centrifuge
Ribosome Binding Sites: A, P, E
III. Translation has 3 Steps, Each Requiring Different Supporting Proteins • Initiation – Requires Initiation Factors • Elongation – Requires Elongation Factors • Termination – Requires Termination Factor
Overview of Prokaryotic Translation
Initiation: 1. Binding of initiation factors to small subunit. 2. Binding of first t. RNA and m. RNA to small subunit. 3. Binding of large subunit.
Elongation: 1. Binding of next t. RNA using EFs at A site. EPA 2. Peptide Bond formation between 2 amino acids. EPA EPA 3. Translocation of ribosome.
Termination: 1. Binding of Release Factor to Stop Codon UGA, UAG. 2. Disassembly
EM of Polyribosomes: >1 Ribosome working on the same m. RNA Rabbit Hemoglobin m. RNA Midgefly Salivary Gland with Nascent Polypeptide Note: occurs in cytoplasm.
IV. Inborn Errors of Metabolism Provided First Evidence that Genes Encode Proteins Alkaptonuria is an inherited disorder first described by Garrod (1902) and Willliam Bateson. – Infants have black urine, darkened ears and nose due to homogentisic acid deposits. – Garrod increased the amino acids phenylalanine and tyrosine in the diet and saw increased deposits in affected individuals only. – He concluded that “unit factors control ferments” (genes control enzymes); results ignored for 30 years.
Phenylketonuria (PKU) • Autosomal recessive human metabolic disorder, first described in 1934. • 1/11, 000 live births, results in mental retardation due to high [Phe] in body fluids. • Homozygotes cannot convert Phe to Tyr, since enzyme phenylalanine hydroxylase is lost. • Treatment: detection in newborns, low Phe diet; prevents mental retardation • Thousands of disorders have been found that result from genetic factors rather than pathogens.
Metabolic Pathways for Phe and Tyr tyrosinase
Other Metabolic Disorders in the Pathway • Albinism – Autosomal recessive – Results from loss of tyrosinase enzyme in skin, which converts Tyr to DOPA and DOPA to Melanin pigments – Loss of tyrosinase in brain causes Parkinson’s Disease (loss of DOPA+ neurons). • Tyrosinemia – Results from loss of tyrosine transaminase
V. Beadle and Tatum: One Gene One Enzyme (Polypeptide) • From mutations in fungus Neurospora • True in many cases, but there are many exceptions: – Some proteins have multiple subunits, each a polypeptide coded by a different gene. – Some genes code for more than one polypeptide, through differential splicing out of introns; e. g. secreted vs. membrane-bound forms of antibody molecules.
- Slides: 24