Announcements

1. Homework - problem set 5 - due this Friday

2. Reading Ch. 14: Skim btm 391 -top 397. Skip rest of 397- 403.

Review of Last Lecture

I. tRNA and the genetic code

II. Transcription - prokaryotes

III. Transcription - eukaryotes

Outline of Lecture 25

I. RNA processing in eukaryotes

II. Translation of mRNA into protein - tRNA 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, mRNA is polycistronic; in eukaryotes, mRNA is usually monocistronic.– Polycistronic: one mRNA codes for more than one polypeptide– moncistronic: one mRNA 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 mRNA in eukaryotes, no processing in prokaryotes

II. Structure: Unusual Bases Found in tRNA

Function of Unusual Bases

• Created post-transcriptionally.

• Purpose is sometimes to allow for promiscuous base-pairing: Inosine in the 1st “wobble” position of anticodon can bind to 3rd U, C or A in codon.

• This means that fewer different tRNAs are required.

• Others play a structural role.

tRNA Structure

Aminoacyl tRNA synthetase

Aminoacyl tRNA Synthetases

• Enzymes which bond specific amino acids to their cognate tRNAs.

• There are 20 different synthetases, one for each amino acid.

• Covalent linkage through an ester bond (amino acid activation) requires ATP.

• tRNA 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 tRNA and mRNA to small subunit.

3. Binding of large subunit.

Elongation:

1. Binding of next tRNA using EFs at

A site.

2. Peptide Bond formation between 2

amino acids.

3. Translocation of ribosome.

E P A

E P A

E P A

E P A

E P A

Termination:

1. Binding of Release Factor to Stop Codon

UGA, UAA, UAG.

2. Disassembly

EM of Polyribosomes: >1 Ribosome working on the same mRNA

Rabbit Hemoglobin mRNA Midgefly Salivary Glandwith 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.