Genetics Ch 13: Translation

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Archibald Garrod

• Propose a relationship between genes and protein production

• Believed that those with alkaptonuria didn’t have the protein to break down the alkapton

Beadle and Tatum

• Mutated Neurospora crassa (bread mold) and found a mutant that couldn’t make the vitamin B6

• A defect in a gene caused a defect in the enzyme that made vitamin B6

1 gene codes for 1 enzyme

• Wiltype bread mold contains 4 different genes that allow it to produce methionine

• 4 different mutant strains of bread mold each missing one gene could not produce methionine

  • Mutant strain 1 is missing gene 1 so it can’t code for the protein to make gene 2 (and so methionine won’t be made)

    • If mutant strain 1 is given gene 1 then it will produce the protein to code for gene 2 and then will produce methionine

A single gene controlled the synthesis of an enzyme

Modifications to Beadle and Tatum’s Findings

• Not all proteins are enzymes

• Some proteins are composed of two or more different polypeptides

• Not all genes code for proteins (functional RNAs)

• One gene can produce multiple proteins through alternative splicing

Start, Stop, and Anticodon

Start codon: AUG

Stop codons: UAA, UAG, UGA

Anticodon: tRNA’s complement to the mRNA codon

Ex: mRNA- UCA tRNA- AGU

Universal and Degenerate

• mRNA code is universal in most cases (ex. UGA is a stop codon universally but codes for something different in mitchondria)

• Multiple codons can code for the same amino acid

Peptide Bonds

• Joining two amino acids together produces water (condensation reaction)

• Beginning of polypeptide chain is N terminus (Amino terminus) and end is C terminus (Carboxyl terminus)

Nonpolar amino acids (11)

Hydrophobic: interior of the folded protein

Glycine

Gly, G, Nonpolar

Alanine

Ala, A, Nonpolar

Valine

Val, V, Nonpolar

Leucine

Leu, L, Nonpolar

Isoleucine

Ile, I, Nonpolar

Proline

Pro, P, Nonpolar

Methionine

Met, M, Nonpolar

Phenylalanine

Phe, F, Nonpolar

Tyrosine

Tyr, Y, Nonpolar

Tryptophan

Trp, W, Nonpolar

Polar and charged amino acids (9)

Hydrophilic: surface of the protein

Serine

Ser, S, Polar Neutral

Threonine

Thr, T, Polar Neutral

Asparagine

Asn, N, Polar Neutral

Glutamine

Gln, Q, Polar Neutral

Aspartic Acid

Asp, D, Polar Acidic

Glutamic Acid

Glu, E, Polar Acidic

Histidine

His, H, Polar Basic

Lysine

Lys, K, Polar Basic

Arginine

Arg, R, Polar Basic

Protein Folding

• Translation produces the primary structure (string of amino acids)

• Protien folding happens during and after translation

Secondary Structure

When hydrogen bonds form between the carbonyl group of one amino acid and the N-H group of another amino acid

• Alpha Helixes: Backbone is coiled

• Beta Pleated Sheets: Segments of chain bend 180 degrees and then fold

Tertiary Structure

• When the protein becomes 3D

• Hydrophobic, ionic, disulfide interactions

• Can have tertiary structures with mainly alpha helixes, beta sheets, disulfide interactions

Quaternary Structure

Interactions between multiple polypeptide chains

Template and Coding Strand

They are complementary
ex. template strand : A T, coding strand : T A

Cell free Translation Systems

• Using homopolymer of RNA to determine genetic code

• Ex: Using RNA or just U’s and observing only one amino acid

Adaptor Hypothesis

Francis Crick believed that tRNA recognized the mRNA codon and brought over the corresponding amino acid

Features of the tRNA

• Three stem loops

• Amino acids covalently attach to the 3’ CCA spot (all tRNA have CCA at its 3’)

• Anti codon to connect to compelemetnary mRNA codon

Charging tRNA molecules

Enzyme aminoacyl-tRNA synthetase charges the tRNA and allows it to bind to the amino acid (20 types of aminoacyl-tRNA synthetase for 20 amino acids)

1. Specific amino acid and ATP bind to enzyme

2. AMP is covalently bound to amino acid and pyrophosphate is released

3. tRNA binds to the enzyme and amino acid is attached to 3’ end

4. Charged tRNA is released

Amino-acetyl tRNA synthetase

• Very accurate and almost always brings the correct amino acid for the correct tRNA

• Recognizes the correct tRNA using its anticodon and other sequences

Wobble Hypothesis

Francis Crick hypothesized that the third position in the mRNA codon could tolerate mismatches which is why there are a limited number of tRNAs but multiple codons can code for one amino acid
Ex: UCC mrna codon has a tRNA anticodon as AGG and codes for serine

UCU mrna codon is almost the same as UCC except for the third position but it also codes for serine; it can tolerate the mismatch between UCU and AGG

Ribosome Structure

• Large and small subunit

• mRNA is between the two sub units

• E site: tRNA exit

• P site: peptide bond formed between amino acids

• A site: where tRNA first enters

• Bacterial cells only have ribosomes in the cytoplasm

• Eukaryotic cells have ribsomes in the cytoplasm and ribosomes in the chrloplasts and mitchondria

Bacterial v Eukaryotic Ribsomes

Svedberg unit (S): the rate at which the ribosome sediments; not additive (ex. bacterial small subunit sediments at a rate of 30S)

Bacteria

• Small Subunit: 30S

  • Contains 16S rRNA

• Large Subunit: 50S

  • Contains: 5S rRNA and 23S rRNA

• Assembled ribosome: 70S

Eukaryotes

• Small Subunit: 40 S

  • Contains 18S rRNA

• Large Subunit: 60 S

  • Contains: 5S rRNA, 5.8S rRNA, 28S rRNA

• Assembled ribosome: 80S

Ribosome Synthesis in Bacteria and Eukaryotes

Bacteria: Occurs in cytoplasm

Eukaryotes: Small and Large subunit are synthesized in the nucleolus and transported to the cytoplasm

Steps of Initiation of Translation in Bacteria

1. mRNA, initiator tRNA, ribosome, and three initiator factors = initiation complex

2. Initiator tRNA is also called tRNA fmet and carries Formal Methionine and binds to Start codon is usually AUG but can be GUG or UUG

3. 16S rRNA binds to the Shine Dalgarno Sequence (upstream of AUG) (5’ UTR on the coding strand of DNA)

   1. Shine Dalgarno sequence in mRNA: UAAGGAGGU (replace U with T to get sequence in DNA coding strand

   2. DNA Coding strand start codon = ATG

4. Initiator factor 1 and Initiator factor 3 bind to the A and E sites of the ribosome; Initiator factor 2 promotes binding of initiator tRNA to P site

5. IF1, IF2, IF3 are released and large subunit is attached; Start codon is positioned into P site with initiator tRNA with fmet

Steps of Initiation of Translation in Eukaryotes

Does not have the Shine Dalgarno Sequence

Eukaryotic Initiation Factor 4 recognizes 5’ cap

• Helps bind mRNA to 40S subunit of ribosome

• Starts the scan for AUG in mRNA (will be connected to initiator tRNA met)

• Kozak Rules: the correct start codon AUG will have either an A or G at the -3 position and a G at the +4 position

• Once AUG is found 40S subunit binds to it and then the 60S joins

Translation Elongation in Prokaryotes

• The 23 rRNA (large subunit) catalyzes the peptide bonds so it is a ribozyme

• The tRNA in the A spot brings in an amino acid

• The 23rRNA catalyzes a peptide bond between the growing protein chain and the new amino acid

• Ribosome is translocated (tRNA moves to E site, new amino acid moves to P site, A site is now open)

Translation Termination Prokaryotes

Terminated using protein release factors; tRNA does not recognize mRNA stop codons (no anticodon)

• RF1: recognizes UAA and UAG

• RF2: recognizes UAA and UGA

• RF3: recognizes no stop codons but still needed in the termination process

Translation Termination Eukaryotes

Terminated using protein release factors; tRNA does not recognize mRNA stop codons (no anticodon)

eRF1: recognizes all stop codons

eRF3: doesn’t recognize any but needed for termination