Single-letter code: M D L Y

The Genetic Code How the genetic code was deduced is quite an interesting but horribly complicated story of prokaryotic genetics. I’ll just give you the Cliff notes version: Francis Crick and Sidney Brenner figured out that: The genetic code maps ‘codons’ of 3 bases into one amino acid. AUA->Ile GAU->Asp AGA->Arg mRNA-> Amino Acid

The Genetic Code Crick and Brenner figured out that: The DNA code is read sequentially from a fixed position in the gene

The mechanism and machinery for translating a protein Three components:

mRNA, rRNA, tRNA and protein synthesis In translation, the language of nucleic acids is translated into a new language, that of proteins mRNA provides the code, in linear digital form, for making a protein tRNA provides an adaptor that links the code in a polynucleotide chain to amino acids that make up the polypeptide chain rRNA and ribosomes provide the decoder. Ribosomes bring together mRNA and tRNA, and catalyze the translation of an mRNA into a polypeptide chain. Ribosomes are the site of protein synthesis. Ribosomes create peptide bonds between amino acids to create proteins

tRNA is the adapter. Complementary to the Codon Amino acid is matched to the Anti-codon.

Two views of the adaptor molecule, transfer RNA (tRNA), which guides amino acids to the mRNA-ribosome complex The anticodon of the tRNA aligns with the codon in mRNA through complementary base pairing 5’ 3’ 5’ 3’

Translation occurs 5’ to 3’ Translation is performed with the help of the Ribosome. Is tRNA orientation with mRNA correct as drawn?

RNA is the major component of the Ribosome. About 2/3 of the Ribosome is RNA by mass. These RNA molecules are called rRNA and they play a central role in the translation of mRNA into polypeptides.

Ribosomal RNAs (rRNAs) form complex 2 o and 3 o structures that are essential for their function What does this rRNA molecule look like in three dimensions?

3-D model of 16s rRNA molecule as it folds in the ribosome… …and overlaid with its protein subunits

Behold, the large subunit containing the 23s + 5s rRNAs Proteins are blue, RNAs are red and white

A E Large subunit P Small subunit Translation - Initiation fMet UAC GAG...CU-AUG--UUC--CUU--AGU--GGU--AGA--GCU--GUA--UGA-AT GCA...TAAAAAA 5’ mRNA 3’

A E Ribosome P UCU Arg Aminoacyl tRNA Phe Leu Met Ser Gly Polypeptide CCA Translation - Elongation GAG...CU-AUG--UUC--CUU--AGU--GGU--AGA--GCU--GUA--UGA-AT GCA...TAAAAAA 5’ mRNA 3’

A E Ribosome P Phe Leu Met Ser Gly Polypeptide Arg Aminoacyl tRNA UCUCCA Translation - Elongation GAG...CU-AUG--UUC--CUU--AGU--GGU--AGA--GCU--GUA--UGA-AT GCA...TAAAAAA 5’ mRNA 3’

A E Ribosome P CCA Arg UCU Phe Leu Met Ser Gly Polypeptide Translation - Elongation GAG...CU-AUG--UUC--CUU--AGU--GGU--AGA--GCU--GUA--UGA-AT GCA...TAAAAAA 5’ mRNA 3’

A E Ribosome P Translation - Elongation Aminoacyl tRNA CGA Ala CCA Arg UCU Phe Leu Met Ser Gly Polypeptide GAG...CU-AUG--UUC--CUU--AGU--GGU--AGA--GCU--GUA--UGA-AT GCA...TAAAAAA 5’ mRNA 3’

A E Ribosome P Translation - Elongation CCA Arg UCU Phe Leu Met Ser Gly Polypeptide CGA Ala GAG...CU-AUG--UUC--CUU--AGU--GGU--AGA--GCU--GUA--UGA-AT GCA...TAAAAAA 5’ mRNA 3’

A E Ribosome P Translation - Termination CGA Phe Leu Met Ser Gly Polypeptide Ala Arg Val CGA GAG...CU-AUG--UUC--CUU--AGU--GGU--AGA--GCU--GUA--UGA-AT GCA...TAAAAAA 5’ mRNA 3’ STOP

A E P Translation - Termination CGA Phe Leu Met Ser Gly Polypeptide Ala Arg Val CGA GAG...CU-AUG--UUC--CUU--AGU--GGU--AGA--GCU--GUA--UGA-AT GCA...TAAAAAA 5’ mRNA 3’ STOP

Translation normally occurs on polyribosomes, or polysomes This allows for amplification of the signal from DNA and RNA, i.e.,… One gene copy Hundreds of mRNAs Thousands of proteins

Translation: the movie

How many bases are required to make a genetic code to serve 20 different amino acids? # of 2-base combos = 4 2 = 16 # of 3-base combos = 4 3 = 64 Not enough! Too many! What is the solution? Evolve a code that is redundant! How? Degeneracy at the third codon position Let’s look at the Genetic Code

Transfer RNA (tRNA) and the genetic code

Many different base modifications occur in tRNA Why are these modifications necessary?

Transfer RNA (tRNA) and the genetic code Legitimate G-U bp Wobble bp

Inosine can pair legitimately with three other bases, when inosine is in the wobble position, i.e., the first, or 5’ base of the anticodon How many chemical changes are required to convert guanine to Inosine? How many chemical changes are required to convert adenine to Inosine?

Is there a logic to the Genetic Code? 8 codon families where the third codon position is fully degenerate, i.e, any of the four bases has the same meaning: (4-fold degeneracy) AC__ ThrGC__AlaCU__Leu UC__SerGG__GlyGU__Val CC__ProCG__Arg Thus, for these 8 amino acids, substituting one base for another at the 3rd position is a SYNONYMOUS change

Is there a logic to the Genetic Code? 7 codon pairs where meaning is the same whichever pyrimidine occurs at 3rd position: (2-fold degeneracy) UAUTyrUGUCys AGUSerAAUAsn UACUGC AGCAAC UUUPheCAUHis GAUAsp UUCCAC GAC 5 codon pairs where purines in the 3rd position are interchangeable: (2-fold degeneracy) AGAArgGAAGluCAAGlnUUALeu AGGGAGCAGUUG AAALys AAG Therefore, half of 3rd position substitutions will be SYNONYMOUS and half will be NON-SYNONYMOUS

This accounts for 17 amino acids: - Trp (UGG) and Met (AUG) are specified by unique codons (non-degenerate codons) - What’s left?Isoleucine (Ile) -- How is Ile specified? (This is one of today’s homework questions) The logic of the Genetic Code 8 families with 4-fold degeneracy = 32 codons 12 families with 2-fold degeneracy = 24 codons + 3 STOP codons (UAA, UGA, UAG) = a 64 total codons

Point mutations Point mutations can affect protein structure and function Type of point mutations: - substitutions (missense and nonsense mutations) - insertions and deletions (frameshift mutations)

Frame Shift Mutations What happens when you get insertions or deletions of bases in the DNA sequence? Usually you end up with a mess. THE BIG FAT CAT ATE THE RAT AND GOT ILL Deletion of one base THE IGF ATC ATA TET HER ATA NDG OTI LL And its all pops and buzzes. Usually frame shift mutations result in premature stop codons.

What is wrong with the mouse on the right? Relationship between genes and proteins Example of a monogenic, or single-gene disorder

Alkaptonuria, aka Black Urine disease, in humans Symptoms: - urine and ear wax turns black as ink shortly after exposure to air -ochronosis: musculoskeletal effects, including progressive degenerative arthritis of the large joints, esp. hip and shoulder - signs of ochronosis: -black deposits in the sclerae (white of eye) -blue colored auricles (ear lobes)

What is the gene defect, or molecular genetic basis, in alkaptonuria? - four point mutations occur in the human HGO gene - Blastp comparison of HGO gene from human and fungus Are any of the sites in these proteins functionally constrained?

Molecular basis of Alkaptonuria Mutations: Proline230Serine and Valine300Glycine account for most cases of the disorder Haplotypes:each of these two mutant alleles constitutes a haplotype, i.e., one haploid type. Progeny inherit one haplotype from each parent.

Categorize three different types of point mutations … ATA TAC GTA CAT … … ile tyr val his … GAAGAA glu … AAA GAA TGG GTT … … lys glu trp val … TGC cys … TCG CCA TGG CCA … … ser pro trp pro… TAGTAG X (stop) 123 Chemical type: Informational type: Functional type: Transversion Transition Missense Nonsense Deleterious Non-synonymous Nondegenerate site Deleterious Non-synonymous Nondegenerate site Deleterious Non-synonymous Nondegenerate site

Categorize three more types of point mutations … ATA TAC GTA CAT … … ile tyr val his … GTC val … AAA GAA TGT GTT … … lys glu cys val … cys … TCG CCA TGG CCA … … ser pro trp pro … A 456 Chemical type: Informational type: Functional type: Transversion Transition Indel (insertion) Missense Frameshift Neutral (silent) Synonymous (4-fold degenerate site) Neutral Synonymous (2-fold degenerate site) Deleterious Non-synonymous TGC … TCG CAC ATG GCC A … … ser his met gly … Is a 4-fold degenerate site ever functionally constrained?

Where can you get more information about the basic concepts embedded within the Central Dogma of Molecular Biology? Here is a great site, full of simple, clear, and animated (!) tutorials: Chapters in this series provides an excellent review of the first group of lectures in Bioinformatics:

Homework #5 : due Monday, Feb. 12 Express fractions as a %

Ribosomes are large ribonucleoprotein (RNP) complexes They are complex affairs, composed of an array of RNA + proteins