602: Genetic Code and tRNAs

Translation

mRNA → Protein

Uses rRNA and tRNA

Translation Ratio

3:1 ribonucleotide:amino acid (CODONS!)

Codons

• Blocks of 3 nucleotides of linear mRNA = 1 amino acid

  • Non-overlapping

  • Three possible reading frames

Genetic Code

• Rules that govern which nucleic acid codon = which amino acid

• Degenerate - some amino acids have more than 1 codon

• Unambiguous - each codon only has 1 aa

Termination Signals

1. UAA (ochre)

2. UAG (amber)

3. UGA (opal)

Start Codon

• AUG (prokaryotes may have others)

  • Methionine - eukaryotes

  • N-formylmethionine - prokaryotes

Other start codons in prokaryotes

AUG (normal), UUG, AUU, GUG

Codon sequence is read…

5’ → 3’

Codon Universality

Evidence that all life arose from a common ancestor

Different Reading Frames

• 1 through 3 on mRNA

  • Amino acid sequence differs between each

Number of reading frames on mRNA

Three

Reading Frames on DNA

• Six:

  • +1, +2, +3 on sense strand (5’ → 3’)

  • -1, -2, -3 on anti-sense strand (3’ → 5’)

Due to having 2 strands

Open Reading Frame (ORF)

Start codon, encoded amino acids, stop codon

Nucleotides must in multiples of 3

How can we predict genes from DNA?

• Look at all 6 reading frames

  • Identify long ORFs and translate them

• Promoters and ribosome binding site help

  • Harder in eukaryotes due to exons/alternative splicing

LONG ORF IS NOT ENOUGH EVIDENCE!

Can a protein be found on all reading frames?

No each is only encoded on a SINGLE reading frame

DNA Mutations

• Permanent changes in DNA code

  • Damage doesn’t always = mutation

Point Mutations

• Changing 1 nucleotide, may/may not affect protein

  • Missense/non-synonymous

  • Silent/synonymous

  • Nonsense

Missense/non-synonymous Mutation

Point mutation that changes 1 amino acid for another

Ex. Sickle cell GAG → GTG = Glu → Val

Silent/synonymous Mutation

Point mutation that DOESN’T change encoded amino acid

Due to degeneracy

Nonsense Mutations

Point mutations that change amino acid → STOP codon

Changes length

Indel

• Insertion/deletion of dNTPs

• To affect reading frame, must be other than multiple of 3

Frameshift mutation

• Indel that alters reading frame because it’s less/more than 3 dNTPs

  • Abnormal sequence

  • Abnormal length

DNA damage = mutation IF:

• DNA damage repair fails

• Mutation is in a coding region (mRNA)/splicing

• Not silent

tRNA

• Contains tertiary structure

• Covalently modified bases

• Charged 3’ amino acid

• mRNA-tRNA base pairing

  • codon-anticodon

• CANNOT recognize STOP codon

mRNA-tRNA base pairing

Complementary base pairing at the wobble position of 5’ anticodon

Acceptor Stem of tRNA

Amino acid binding site (5’ end, charged)

Anticodon loop in tRNA

mRNA binding site for complementary base pairing

Uridine Modifications

• Dihydrouridine (D)

• Pseudouridine

• Ribothymidine

Dihydrouridine

Lacks a double bond, found in D-loop

Pseudouridine

Ribose is attached to C5 of uridine instead of N1, in TWC loop

MOST COMMON modification

Ribothymidine

Methylated uridine, in TWC loop

Adenosine Covalent Modification

Deaminated → INOSINE, found in wobble position

Can pair with A, C, U

Wobble Hypothesis

• If tRNA wobbles, it can non-Watson-Crick base pair

  • 5’ anticodon with 3’ codon

• G = U

• Inosine (anticodon) — A, C, U

• One charged tRNA can recognize MULTIPLE codons

Inosine can wobble with…

A, C, and U due to covalent modification

Which bases can’t wobble in anticodon?

A and C

Charged tRNA

Contains charged amino acid from aminoacyl-tRNA synthetase

Aminoacyl-tRNA Synthetases

• FIRST step in protein synthesis

• Amino acid bound to tRNA acceptor stem

  • 2 steps, requires ATP

Class I Transfer

Amino acid attached to 2’ OH of tRNA, quickly moved to 3’ OH

Class II Transfer

Amino acid attached to 3' OH of tRNA

Proofreading of tRNA Synthetases

• Based on amino acid and tRNA properties

  • Wrong side chain = hydrolysis (shape/size/property/anticodon)

• Cuts amino acids off tRNA

Proofreading before synthesis:

Hydrolyzed intermediated

Proofreading after synthesis:

Hydrolyzed Product