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Translation Basics

• Takes place in the cytoplasm '

• Translating the coding region of the mRNA by the ribosomes

How do you make a Protein?

Amino Acids (AA) are building blocks

• Peptide bonds link AA’s together

• They have a carboxyl group and an amino group

• Synthesis starts at the animo end

• Synthesis ends at the carboxyl end

• All polypeptides have an amino and carboxyl terminus

• Polypeptides are made of many amino acids linked together by peptide bonds

• Proteins are chemically distinct from one another due to differences in the R-groups

• A proteins consists of one or more polpeptides proteins function results from the 3D shape and chemical properties of the amino acids

Ingredients of Translation

1. mRNA (the instructions) with codons (the words)

2. Loose amino acids (raw material)

3. tRNA- charged (amino acids attached)

   1. Aminoacyl tRNA synethetases- attached AA to the correct tRNA

4. Ribosomes

   1. Initiation factors

   2. elongation factors

   3. peptidyl transferase

5. Energy (ATP and GTP)

6. release factors

Features of the genetic code

• Read codon by codon (3 nucleotides )

• A codon specifies the amino acid to be added- each codon translates to one amino acid

• Degenerate (redundancy)- 1 AA can be coded for by more than 1 codon- the code has more info than needed 61 codons=20 AAs

• Start and stop codoons- Start : AUG (Met or fMet); Stop: UAA,UGA,UAG

• There are no pauses in the code (every codon is read)

• Universal- same code across life

Reading Genetic Code

“The Wobble”

• Often the 3rd position of codon can be any nucleotide where 1st and especially 2nd are “stable”

• Groups of codons that code for the same AA are called synonymous Z

Amino Acids

• R groups in Amino Acids can make the molecules:

  • Non-polar( hydrophobic)- No charge, no H bonds, repel water

  • Polar (hydrophilic)- Charged, H-bonds, bonds with water

  • Positive charge (+)

  • Negative charge (-)

tRNA- Charging

Aminoacyl tRNA Synthetases: direct the charging of each tRNA (20 different synthetases to match AA with correct tRNAs

Degeneracy of the Code if tRNA

• Multiple tRNAs can carry the same Amino Acid

• Different codons sometimes pair with the same anticodon→ Wobble

  • Flexibility in base pairing

  • Many codons differ at only the third base

The Ribosomes (rRNA and Proteins)

• rRNA makes up ~60% of the ribosome, the ribosomes contains proteins that help the rRNA fold into the right shape

Steps in Translation

1. Initiation: Assemvle the ribisomoes on the mRMA postiioned correctly to find the START codon bring in the first Amino Acid

2. Elongation: A cyclical prpocess of bringing in the nect amino acoid and catalyzing peptide bonds to grow the amino acid chain (the polypeptide).

3. Termination: When the STOP codon is reached, bring in release factors and break down the assembled components– releasing the completed polypeptide.

Initiation In Bacteria

Requires:

• mRNA

• Charged fMet (euk) and Met (prok)

• Ribsome: large and small subunit

• Initation factors : IF1 IF2 IF3

• Energy GTP

First:

• The pre-initiation comeplex: the small subunit with IF3 ninds near the 5’ end of the mRNA and searched for the Shine-Dalgarno Sequence

  • Shine paris with the small subinit to position the start codon in the P site (only initiationor with tRNA can go to thw p site- every onther amino acid goes to the A site)

• The tRNA charged with fMet forms a complec with IF2 and GTP and binds to the start codon on the p Site through pairing of the anticodon and codon

  • IF1 binds to the A site and blocks it

• GTP provides energy to jpiin thew small and large subunit

• IF1 is released for the A site and charged tRNA can now enter the site

• IF3 is released and its now ready to buildf the polypeptide

Initiation in Eukaryotes

1. Cap binding proteins binf to the mRNA 5’cap and form a closed loop structure with the poly a tail

2. eIFs recruit the small subunit and prevent binding of the large subunit

   1. tRNA met binds to the P site

3. Complex scans for the start codon (AUG) in tjhe kozak sequence

4. Finding AUG triggers binding of the large subunit (60S)

Elongation in Bacteria

1. Happens in the ribosomes- Charged tRNAs enetr the A site

2. Peptide bond forms betwen the AA at the P site and AA at the A site. Growing Aa chain (polypeptide) is transferred to the A site

3. tRNA that is released the polypeptide that was in the Psite is moved to the E site which means it exits the ribosome

A Site

1. Elongation factor EF-Tu and GTP attach to a charged tRNA → this provides energy for the next step

2. Many charged tRNAs could enter the A site

3. Peptidyl Transferase ribozyme catalyzes:

   1. Hydrolisis of bond vetweent tRNA and AA (p site)

   2. Formation of a peptide bond between two Amino Acids

Translocation

1. Elongation factor EF-G + GTP (energy) binds to the A site which pushes everything over → dont have to know that

2. nn

Termination (in Bacteria)

1. RF1 recognizes UAA or UAG, or RF2 recognizes UGA or UAA

2. Then RF3 binds GTP and the ribosome

3. The polypeptide chain is cleaved from the tRNA

4. Result is confromational shape change

Differences

• Prokaryotic: concurrent transcription and translation

  • Initiation- shine delgarn sequence

• Eukaryotic: Transcription and translation separated in time and space

  • Initiation- 5’ cap and Kozak sequence

After Translations:

• Folding

  • Primary, secondary, tertiary. etc structure

• Chemical modification

• Trimming of the methionine

Mutations

• Single nucleotide change= Point Mutation (type of Misense mutation)

• Synonymous mutation (silent/neutral) nucleotide change which causes no effect die to changed a codon to a synonymous codon (Silent mutation)

• Nonsense Mutation- a nucleotide change that results in a STOP codon during translation

• Frameshift Mutation- the gain or loss of nucleotides resulting in a change in the reading frame during translation (deletion or insertion)