RNA to Protein

What is translation

converting the mRNA sequence into the language of amino acids  

• Follows “genetic code” rules 

• RNA copies of DNA segments direct synthesis of protein 

• Used by ALL present-day organisms (except the mitochondria) 

• Requires adaptor molecule (tRNA) 

What is an anticodon

a three-nucleotide sequence on a tRNA molecule that is complementary to a specific codon on mRNA

What is a codon

a sequence of three consecutive nucleotides (base pairs) in mRNA that codes for a specific amino acid or signals the termination of protein synthesis

• Read in mRNA 

• First 2 positions of the codon MUST match with the anticodon correctly (3^rd can be mismatched) 

• 64 possible combinations of codons, but 20 amino acids 

What are eukaryotic initiation factors

Protein that helps load initiator tRNA on to the ribosome, thus initiating translation (eIF)

What is the genetic code

set of rules followed during translation 

• Ex: AAA = Lys or UGC = Cys 

• Redundant because of the wobble on the 3^rd base 

• Variations: 

  • Translation recoding = 21^st amino acid can be inserted directly into growing polypeptide using selenocysteine method 

    • Seleoncysteine = produced from serine tRNA that is enzymatically altered 

      • SC tRNA binds to STOP codon, translating the codon into SC 

  • Translational frameshift = similar to leaky scanning (allows more than one protein to be synthesized from a single mRNA by changing the reading frame by ignoring a start or stop codon) 

    • Tends to happen in viruses only 

What is wobble

3^rd position in a codon that doesn’t need to match with the anticodon 

• Runs a risk of mutation 

Wobble codon base	Possible anticodon bases
U	A, G, or I
C	G or I
A	U
G	C

What is an initiator tRNA

the first tRNA that begins translation at AUG codon 

• All new polypeptide chains start with MET (from initiator tRNA)  

  • AA is eventually removed by protease 

What is the reading frame

allow an RNA sequence to be translated in any one of three different frames  

• Only one of the three is an actual mRNA that encodes for the required protein 

  • Depends on where the frame starts 

• Start point is crucial to determine whole reading frame 

What is the proteasome (simple)

 a cylinder that contains proteases (inside) for digesting proteins 

What is the ribosome

made of 50 different ribosomal proteins and several rRNAs 

• Made of 2 subunits 

  • 1 large = catalyzes the formation of peptide bonds between amino acids 

  • 1 small = important for matching tRNA to codon on mRNA 

    • Matches anticodon to codon 

• Adds the amino acids to the polypeptide chain 

What is a ribozyme

catalytic activity found in RNA molecules 

• rRNA is responsible for catalytic activity in forming covalent peptide bonds (NOT PROTEINS) 

• Responsible for catalytic activity by donating an OH to the active site when forming covalent peptide bonds (NOT Proteins) 

What is a tRNA

Small set of RNA molecules that acts as an adaptor molecule during translation 

• 80 nucleotides long & synthesized by RNA pol. III in nucleus 

  Splicing process has NO lariat formation (only cut and paste) 

• ~50 different tRNAs because of chemical modifications of bases 

  Not one per amino 

• Responsible for catalytic activity by donating an OH to the active site when forming covalent peptide bonds (NOT Proteins) 

• Each tRNA is unique and is synthesized by a AA-tRNA synthetase  

  Ensures proper matching of anticodon to amino acid 

• 2 critical regions of unpaired nucleotides 

  Anticodon = complementary to mRNA codon 

  Amino acid attachment site at 3’ end 

What is ubiquitin

highly conserved eukaryotic protein with many functions (ex: serves as a marker for proteasomes) 

What is a nonsense mutation

Premature stop mutation 

What is a missense mutation

A different amino acid at a different location  

What is a start codon

AUG (Met) bound to tRNA (required to begin translation) 

What is a stop codon

 UAA, UAG, UGA 

What is a ribosome and it’s function

Add the amino acids to the polypeptide chain 

• Has 4 binding sites for RNA 

  1 for mRNA 

  A & P site for tRNAs 

  • Hold tRNA tightly if successfully found a complementary anti-codon to codon  

  • Sites are close together in order to avoid changing the readframe (tRNAs stay next to each other) 

  E site to exit the tRNA 

• 4 major steps to chain elongation: 

1. tRNA binding 

2. Peptide bond formation 

3. Large subunit translocation 

4. Small subunit translocation 

What is the process of adding an amino acid to a tRNA

tRNA synthetase: 

• tRNA requires aminoacyl-tRNA (AA-tRNA) synthetase to recognize and attach the correct amino acid on the 3’ end 

  20 diff. AA-tRNA synthetases – one for each specific amino acid 

  Reaction coupled with ATP hydrolysis 

  • Uses ATP to hydrolyze bonds and add the correct amino acid 

• Must recognize the matching tRNA anticodon 

  Contains 3 adjacent nucleotide binding pockets (each with complementary shape and charge to anticodon nucleotide) 

  Several positions on tRNA are important for synthetase recognition (ex: D loop) 

Two editing mechanisms that ensures the correct amino acid to tRNA synthetase 

1. Active site on enzyme has high affinity for the appropriate amino acid 

2. A second proofreading mechanism moves bound amino acid into an editing site (if it fits, it’s rejected by the editing site) 

Describe the translation process

• Appropriate start sequence, necessary to determine reading frame 

  • With special initiator tRNA at AUG 

• Initiation factors recognize initiator tRNA, allowing synthesis to begin 

Steps

1. Initiator tRNA (MET) binds to P-site on small subunit ribosome with the help eIF

2. Small ribosome binds to 5’ end of mRNA (has it’s own eIFs)

3. Small ribosome moves along mRNA until it finds an AUG

   • driven by ATP

   • leaky scanning (helps find correct AUG)

4. eIF2 leaves small ribosome. Allowing it to bind to the large ribosomal subunit (P with MET and free A site)

5. AA-tRNA binds to A site and protein synthesis begins (peptide synthesis steps)

Peptide synthesis

1. Aminoacyl-tRNA molecule binds to vacant A site (if codon and anticodon match) 

   • P-site is bound by another tRNA 

   • AA-tRNA binds to mRNA codon in the A-site 

   • mRNA translated in 5’ to 3’ direction (starting with amine (N) terminus) 

     • Amino acids added at C terminus 

2. Carboxyl end of polypeptide chain is released from P site tRNA and new polypeptide bond is formed 

3. Large subunit translocates relative to the small subunit 

   • Sites are hybrid  

   E & P on large subunit 

   P and A on small subunit 

4. Small subunit translocates (RESET RIBOSOME) 

   • Moving the mRNA 3 nucleotides to the A-site  

What is the amino acid chain forming

• Protein chain made N-terminal to C-terminal end 

• Peptide bonds formed between the carboxyl group at the end of the growing peptide chain and free amino group on incoming amino acid 

  • Growing carboxyl end still covalently attached to tRNA (bond energy used to attach next AA-tRNA) 

What are the steps for translation initiation?

1. initiator tRNA (MET) binds to P-site on small subunit ribosome with the help eIF

2. Small ribosome binds to 5’ end of mRNA (has it’s own eIFs)

3. Small ribosome moves along mRNA until it finds an AUG

   • driven by ATP

   • leaky scanning (helps find correct AUG)

4. eIF2 leaves small ribosome. Allowing it to bind to the large ribosomal subunit (P with MET and free A site)

5. AA-tRNA binds to A site and protein synthesis begins (peptide synthesis steps)

How is translation stopped?

Once a STOP codon is reached (UAA, UAG, or UGA) = they DON’T code for an amino acid 

• When STOP codon is at the A site, it calls over release factors to enter the rRNA and force the enzymes in the ribosome to add water (NOT an amino acid) 

  • Forms COOH end (C terminus) 

• The release factor = molecular mimic (similar structure to tRNA => helps release factor bind to A site) 

peptide chain is released and begins further folding

8. Newly transcribed mRNA strand is moved through many ribosomes before degradation 

• Allows for a lot of proteins to be made from 1 transcript 

9. Protein folding happens immediately as it is being made 

What are the steps of peptide chain synthesis?

1. Aminoacyl-tRNA molecule binds to vacant A site (if codon and anticodon match) 

   • P-site is bound by another tRNA 

   • AA-tRNA binds to mRNA codon in the A-site 

   • mRNA translated in 5’ to 3’ direction (starting with amine (N) terminus) 

     • Amino acids added at C terminus 

2. Carboxyl end of polypeptide chain is released from P site tRNA and new polypeptide bond is formed 

3. Large subunit translocates relative to the small subunit 

   • Sites are hybrid  

   E & P on large subunit 

   P and A on small subunit 

4. Small subunit translocates (RESET RIBOSOME) 

   • Moving the mRNA 3 nucleotides to the A-site  

What is proofreading

Elongation factors (EF1/2) check tRNA/amino acid pairing for accuracy by providing GTP 

• • If it’s not the correct tRNA, then it will not provide energy (GTP) 

• & speed up translation process 

  • Helps bring tRNA to the ribosome 

• Codon-anticodon match checked with GTP hydrolysis (99.99% accuracy) 

  • Small rRNA subunit forms hydrogen bond between the codon and anticodon 

    • If it's a correct match, GTP hydrolysis happens. Moving translation process forward 

    • If it's an incorrect match, tRNA leaves the ribosome 

Quality control during translation:  

1. 5’ cap and poly A tail = essential for translation initiation 

2. Exon junction complex ensures proper splicing (essential) 

3. Nonsense-mediated mRNA decay (MOST POWERFUL) 

   • When there is more mRNA left to translate but a STOP codon has already been reached (ribosome “senses” there are more EJC therefore, mRNA is destroyed) 

   • Never gets translated into a protein 

What are some diseases caused by ribosomopathies

a collection of rare disorders involving impaired ribosome biogenesis and function (mutations affect translation and protein synthesis) 

Ex: Diamond Blackfan Anemia, Dyskeratosis Congenita, & Treacher Collins 

• Alterations in genes encoding ribosomal proteins 

  • Diamond-Blackfan Anemia = congenital mutation in RPS 19 causing hypoplastic macrocytic anemia & bone marrow failure 

• Alterations in genes involved in ribosomal biogenesis 

  • Myelodysplastic syndrome = congenital mutation similar to DB anemia 

  • Treacher-Collins syndrome = cartilage hair hypoplasia 

• Ribosomal function abnormalities activate p53 pathway 

  • 5q syndrome = entire q arm of chromosome 5 deleted. Presents with severe macrocytic anemia 

What are some diseases caused by nonsense mutations

Rare. Caused by truncated protein because STOP codon is misplaced 

• Some cystic fibrosis mutations 

• DMD 

• Beta-thalassemia 

• Hurler’s syndrome 

PTC 124 drug was in clinical trials = makes ribosomes less sensitive to STOP codons 

What are some diseases caused by missense mutations

Different amino acid at a different location causes... 

• Sickle cell disease 

• Some types of cystic fibrosis mutations 

• Some cancer types