Ch 13.1 Translation

History, Genetic Code, tRNA

___________

• noticed that pts with ________ (a genetic condition) lacked enzyme _________

→ proposed that patients with diseases lacked an enzyme

→ connected concept of _____ to _____

Archibald Garrod

• noticed that pts with alkaptonuria (a genetic condition) lacked enzyme homogentisic acid oxidase

→ proposed that patients with diseases lacked an enzyme

→ connected concept of genes to enzymes

enzyme mutations → problems in intermediates of metabolic pathways → human diseases

(view phenylalanine metabolism for concept)

_________ & _________

• purposefully created mutations to verify mutations followed Mendelian inheritance

• studied _________ (type of mold, fungi)

  • used x-rays to damage DNA

• looked for cells lacking enzymes in the pathway to produce amino acids, including:

  • _________

  • _________

• concluded __________________ hypothesis

Beadle & Tatum

• purposefully created mutations to verify mutations followed Mendelian inheritance

• studied Neurospora crassa (type of mold, fungi)

  • used x-rays to damage DNA

• looked for cells lacking enzymes in the pathway to produce amino acids, including:

  • arginine

  • methionine

  • concluded one gene-one enzyme hypothesis

Beadle & Tatum grew plates with minimal media. They added substrates one at a time based on the pathway. For example, methionine:

1. __________

2. __________

3. __________

4. __________

5. Methionine

Beadle & Tatum grew plates with minimal media. They added substrates one at a time based on the pathway. For example, methionine:

1. Homoserine

2. O-acetylhomoserine

3. Cystathionine

4. Homocysteine

5. Methionine

View practice question from HW (1)

HW 7 Q13

• we want to “rescue” bacteria by giving them products that can get them arginine

• so, all grow if given arginine

• most grow if given second last product (unless very last enzyme is mutated)

• and so on…

View practice question from HW (2)

HW 7 Q14

• if almost everything can grow, early enzyme was mutated

• if nothing can grow, late enzyme is mutated

One Gene-One Enzyme Hypothesis = one gene controls synthesis of one enzyme

• 4 modificatiosns were made to this theory

  1. enzymes are only one category of ________

  2. some proteins are made of 2+ ________

  3. some genes code no polypeptides

     • ex: _______

  4. some genes can code many polypeptides

     • via ___________

One Gene-One Enzyme Hypothesis = one gene controls synthesis of one enzyme

• 4 modificatiosns were made to this theory

  1. enzymes are only one category of proteins

  2. some proteins are made of 2+ polypeptides

  3. some genes code no polypeptides

     • ex: tRNA, rRNA, etc.

  4. some genes can code many polypeptides

     • via alternative splicing

one gene-one enzyme hypothesis was later updated to __________ hypothesis

one gene-one enzyme hypothesis was later updated to one gene-one polypeptide hypothesis

The Genetic Code = mRNA → amino acids

• almost universal code

• ____ possible codons

• start codon: ___

  • codes amino acid _________

• stop codons: ___/___/___

The Genetic Code = mRNA → amino acids

• almost universal code

• 64 possible codons

• start codon: AUG

  • codes amino acid methionine

• stop codons: UAA/UAG/UGA

the genetic code has _________ = multiple codons can specify same amino acid

• __________ codons = codons that specify same amino acid

the genetic code has degeneracy = multiple codons can specify same amino acid

• synonymous codons = codons that specify same amino acid

Exceptions to the genetic code

1. __________ (___) = “21st amino acid”

   • codon = ___

2. __________ (___) = “22nd amino acid”

   • codon = ___

• downstream sequences are needed in order to incorporate these

• found in specialty enzymes

Exceptions to the genetic code

1. selenocysteine (Sec) = “21st amino acid”

   • codon = UGA

2. pyrrolysine (Pyl) = “22nd amino acid”

   • codon = UAG

• downstream sequences are needed in order to incorporate these

• found in specialty enzymes

______________ = sequence of codons beginning with start codon

• _______/_______ can cause reading frame shift → amino acid sequence change

reading frame = sequence of codons beginning with start codon

• deletions/insertions can cause reading frame shift → amino acid sequence change

During polypeptide synthesis, a __________ bond is formed between old C/N-terminus and new C/N-terminus

During polypeptide synthesis, a peptide bond is formed between old C-terminus and new N-terminus

4 levels of protein structure

1. Primary = amino acid sequence

   • determines how protein folds

   • folding may be aided by _________

2. Secondary = _________ and _________

   • stabilized by _________ in peptide backbone

3. Tertiary = 3D structure

   • determined by _________, _________, _________ forces and _________

4. Quaternary = 2+ polypeptides → fully functional protein

4 levels of protein structure

1. Primary = amino acid sequence

   • determines how protein folds

   • folding may be aided by chaperones

2. Secondary = alpha helix and beta sheets

   • stabilized by H-bonds in peptide backbone

3. Tertiary = 3D structure

   • determined by hydrophobic, ionic, van der Waals forces and H-bonds

4. Quaternary = 2+ polypeptides → fully functional protein

Note!!! Review amino acids!!!

Proteins have many functions:

• _________ - ex: sodium channels, hemoglobin

• _________ - ex: myosin

• _________ - ex: tubulin

• _________ - ex: insulin, insulin receptor

• _________ - ex: integrins

Proteins have many functions:

• transport - ex: sodium channels, hemoglobin

• movement - ex: myosin

• cell shape & organization - ex: tubulin

• cell signaling - ex: insulin, insulin receptor

• cell surface recognition - ex: integrins

Note: every protein has a unique structure to match its function

Enzymes review…

Experimental Determination of Genetic Code

• deciphered by many researchers including _________ & _________

• they used a ____________ system to produce amino acids, contains:

  • ribosomes, tRNAs, enzymes, etc.

• they ________ amino acids to make them easy to detect

Experimental Determination of Genetic Code

• deciphered by many researchers including Nirenberg & Khorana

• they used a cell-free translation system to produce amino acids, contains:

  • ribosomes, tRNAs, enzymes, etc.

• they radiolabeled amino acids to make them easy to detect

__________ = linked ribonucleotides into RNA → made ______ RNA

• does not require template, order of nucleotides is random

• an experimenter can control amount of nucleotides added and determine the codons by percentages of amino acids

  • ex: 70% G and 30% U

polynucleotide phosphorylase = linked ribonucleotides into RNA → made synthetic RNA

• does not require template, order of nucleotides is random

• an experimenter can control amount of nucleotides added and determine the codons by percentages of amino acids

  • ex: 70% G and 30% U (view image)

Genetic Code Experiment Details

1. Put ___________ into 20 tubes

Genetic Code Experiment Details

1. Put cell-free translation system into 20 tubes

Genetic Code Experiment Details

2. Add ________ molecules

2. Add random mRNA molecules of G and U (made with polynuc phosphorylase)

Genetic Code Experiment Details

3. Add a different _________ amino acid to each tube + 19 other _________ amino acids

Genetic Code Experiment Details

3. Add a different radiolabeled amino acid to each tube + 19 other unlabeled amino acids

Genetic Code Experiment Details

4. ________ for 60 minutes for translation to occur

Genetic Code Experiment Details

4. Incubate for 60 minutes for translation to occur

Genetic Code Experiment Details

5. Add __________ which precipitates ________

Genetic Code Experiment Details

5. Add tricholoracetic acid (TCA) which precipitates polypeptides, not amino acids

• polypeptides => has been translated

Genetic Code Experiment Details

6. Capture precipitated polypeptides in a _____

7. Count radioactivity on filter in a _________

8. Calculated amount of radiolabeled amino acids in precipitated polypeptides

Genetic Code Experiment Details

6. Capture precipitated polypeptides in a filter

7. Count radioactivity on filter in a scintillation counter

8. Calculated amount of radiolabeled amino acids in precipitated polypeptides

Interpretation of Experiment (just read)

• ex: 49% glycine was found

  • from 70% G and 30% U, we know there is a 34% chance of GGG and 15% GGU, which adds up to 49% glycine

  • so, we can conclude that GGG and GGU most likely code for glycine

• multiple experiments were needed to reveal more about the entire genetic code

__________ = RNA molecule made up of multiple nucleotides in a pattern

• ex: UCUCUCUCUC

  • can form codons ___ and ____

• these were used in above experiment to crack the genetic code

• created by Khorana by creating short RNAs (2-4 nucleotides long) with a defined sequence and linking them

RNA copolymers = RNA molecule made up of multiple nucleotides in a pattern

• ex: UCUCUCUCUC

  • can form codons CUC and UCU

• these were used in above experiment to crack the genetic code

• created by Khorana by creating short RNAs (2-4 nucleotides long) with a defined sequence and linking them

Additional Experiment: Triplet-Binding Assay

• __________ and __________ discovered that 3 nucleotides in RNA directly corresponded to amino acids

• so, they conducted another experiment to obtain the genetic code with clearer patterns

Additional Experiment: Triplet-Binding Assay

• Nirenberg and Leder discovered that 3 nucleotides in RNA directly corresponded to amino acids

• so, they conducted another experiment to obtain the genetic code with clearer patterns

Triplet binding assay

1. They mixed one __________ in 20 different tubes with a different radiolabeled amino acid

Triplet binding assay

1. They mixed one triplet RNA in 20 different tubes with a different radiolabeled amino acid

Triplet binding assay

2. Samples were filtered - only amino acids stuck to _______ were retained

→ showed which amino acid this triplet RNA corresponded to

Triplet binding assay

2. Samples were filtered - only amino acids stuck to ribosome were retained

→ showed which amino acid this triplet RNA corresponded to

Structure and Function of tRNA

—

__________ hypothesis (by Crick) = tRNA has 2 main functions:

1. recognize codon in mRNA

2. carry amino acid based on codon

adaptor hypothesis (by Crick) = tRNA has 2 main functions:

1. recognize codon in mRNA

2. carry amino acid based on codon

tRNA are named by amino acid they carry

• ex: ______ = tRNA that carries phenylalanine

tRNA are named by amino acid they carry

• ex: tRNA^Phe = tRNA that carries phenylalanine

tRNA structure

• 3 ____-loop structures

• 3’ ____ acceptor site

• a few _________ sites (differ between tRNAs)

• modified nucleotides

  • I = ______

  • ml = ______

  • T = ______

  • UH₂ = ______

  • m₂G = ______

  • P = ______

tRNA structure

• 3 stem-loop structures

• 3’ ACC acceptor site

• a few variable sites (differ between tRNAs)

• modified nucleotides

  • I = inosine

  • ml = methylinosine

  • T = ribothymidine

  • UH₂ = dihydrouridine

  • m₂G = dimethylguanosine

  • P = pseudouridine

Charging tRNAs

• ______________ = attach amino acids to tRNAs ( = “charging”)

  • there are 20 of these (one per aa)

• charged tRNA = ______________ = tRNA with attached aa

Charging tRNAs

• aminoacyl-tRNA synthetases = attach amino acids to tRNAs ( = “charging”)

  • there are 20 of these (one per aa)

  • charged tRNA = aminoacyl-tRNA = tRNA with attached aa

Charging tRNAs Mechanism

1. _________ & _________ bind to enzyme

Charging tRNAs Mechanism

1. amino acid & ATP bind to enzyme

Charging tRNAs Mechanism

2. ____ is released

Charging tRNAs Mechanism

2. PPi is released

Charging tRNAs Mechanism

3. _______ binds to enzyme and ____ is released

Charging tRNAs Mechanism

3. tRNA binds to enzyme and AMP is released

Charging tRNAs Mechanism

4. charged tRNA is released

Charging tRNAs Mechanism

4. charged tRNA is released

Additional Notes

• aminoacyl tRNA synthetases are very ______!

  • = “second genetic code”

• tRNA has sequences used as _______ sites

• _________ may affect

  • translation rates

  • recognition by aminoacyl tRNA synthetases

  • codon-anticodon recognition

Additional Notes

• aminoacyl tRNA synthetases are very accurate!

  • = “second genetic code”

• tRNA has sequences used as recognition sites

• modified bases may affect

  • translation rates

  • recognition by aminoacyl tRNA synthetases

  • codon-anticodon recognition

genetic code usually has degneracy at ___ position

• exceptions: serine, leucine, arginine (these have a different reason for wobble)

genetic code usually has degneracy at 3rd position

• exceptions: serine, leucine, arginine (these have a different reason for wobble)

• for example… (view image)

__________ hypothesis (by Crick) =

When codons are being paired with anticodons…

• the first 2 positions pair ________ with AU/GC rule

• the 3rd position moves a little = “______”

  • → tolerates mismatches

Wobble hypothesis (by Crick) =

When codons are being paired with anticodons…

• the first 2 positions pair strictly with AU/GC rule

• the 3rd position moves a little = “wobbles”

  • → tolerates mismatches

________ tRNA = different codons, same aa

• these may recognize same codon due to wobble

isoacceptor tRNA = different codons, same aa

• these may recognize same codon due to wobble

• ex: for serine

  • tRNA 1 with anticodon AGA → codon UCU

  • tRNA 1 with anticodon AGA → codon UCU, UCU via wobble

Wobble pairing rules (idk if I need to know, view image)