BIOL 3000 Translation

Translation

Forming a polypeptide chain (protein)

Where: Cell cytoplasm (eukaryotes) associated with ribosomes. Out of the nucleus

When: Either G1 or G2 (the period where genes coding for cellular organelles are synthesized)

Why do transcription and translation mostly occur during G1 and G2?

There always has to be proteins being made

What ribosome does translation depend on?

80S Ribosome

Ribosome

Highly complex cellular machine responsible for the synthesis of proteins from mRNA

Originally called “Palade Particles”

Contains the A, P, E sites, 60S, and 40S subunits

Function of ribosomes

Translates mRNA into peptide chains (protein biosynthesis)

80S Ribosome

A complex of proteins and ribosomal RNAs

The proteins bring the function, the ribosomes bring the structure

60S Ribosome

Large subunit that joins the amino acids together to form a peptide chain

x3 rRNA molecules and 46 proteins

40S Ribosome

Small subunit that is responsible for reading the mRNA 5’ → 3’

x1 rRNA molecules and 33 proteins

Function of 40S subunit

It will search for the mRNA by looking for the 5’ cap. Once recognized, the large subunit will be recruited.

A Site

Aminoacyl-tRNA binding site

The first site that tRNA goes into for translation

P Site

Peptidyl-tRNA binding site

Where the growing chain is held

E Site

Exit site

The site where tRNA leaves

tRNA

Transfer RNA

The RNA molecule that serves as the physical link between mRNA and amino acids

Outside in the cytoplasm associating with amino acids

Contains: Psi (T) arm, D arm, variable arm, and anticodon arm

Psi (T) Arm

Provides structural purpose to the tRNA

Variable arm

Identifies which tRNA interacts with which amino acid

D Arm

Provides structural purpose to tRNA

Anticodon Arm

Interact with the complement mRNA

If GCC, then CGG

Codon

Three bases in an RNA molecule

3 nucleotides that are read at a time from 5’ to 3’

3 nucleotides = ?

1 codon

1 codon = ?

1 amino acid

Start Codon

AUG

Stop Codons

UAA

UGA

UAG

Reading frame

Refers to the one of three possible ways of reading a nucleotide sequence. It is identified based on where we start

We can change the reading frame because not ALL AUG are start codons; however, every stop codon is a stop codon.

Open reading frame

A sequence of DNA triplets, between the initiator and terminator codons, that can be transcribed into mRNA and later translated into protein

mRNA

Genetic code

Written in linear form

Comma-less (no pauses)

Coded in triplets (codons)

Unambiguous (1 codon = 1 amino acid)

Degenerate (1 amino acid can be coded by more than one codon)

Specific start and stop codons

Non-overlapping

Universal

Players in Translation

1. Ribosome (construction site)

2. mRNA (blueprint)

3. tRNA (delivery system)

Steps of Translation

1. Activation

2. Initiation

3. Elongation

4. Termination

Activation

• The correct amino acid covalently bonds to the correct tRNA (charging)

• tRNAs bring the correct amino acid to the ribosome to build polypeptide chains

• Aminoacyl-tRNA synthetase catalyzes the reaction using ATP

• Free amino acids are covalently attached to the 3’ end of the specific tRNAs (non-anti codon end)

• Recognition features in the structure of tRNA determine which amino acid binds

• Some tRNAs can bind multiple amino acids due to third position “wobble”

Aminoacyl-tRNA synthetase

Synthesizes amino acids onto tRNA

Initiation

• Ribosome assembles and binds to mRNA molecule

• The 40S subunit looks for mRNA with the 5’ guanine cap to associate with the mRNA molecule

• The 40S subunit binds to an aminoacyl-tRNA synthetase containing Met and UAC anticodon

• It then recruits the 60S ribosome to form the 80S ribosome

• The MET-tRNA occupies the P site of the 80S ribosome, only when the 60S is recruited

Elongation

• Sequential binding of the aminoacyl-tRNA to the ribosome along with GTP and elongation factors (EF)

• After charged tRNA is placed in the A site, phosphate is released from the GTP, providing energy.

• The peptide bond forms between amino acids in P and A sites, then the amino acid in the P site is released from the tRNA

• The ribosome moves down the mRNA strand (5’→3’) through translocation

• The tRNA that was in the P site moves to the E site to be released into the cytoplasm; tRNA with growing peptide chain moves to the P site, then the A site is vacant and ready for the next charged tRNA allowed to enter

Elongation factors (EF)

Allows specific entry of an incoming tRNA molecule at the A site of the small ribosomal subunit with GTP (energy).

The cleavage of GTP to GDP releases elongation factors

EF-Ts (EF-1B in eukaryotes) acts as a Guanine Exchange Factor (GEF) to exchange GTP in place of the GDP on EF-Tu

Initiation factors (IF)

Associated with the A site to prevent the wrong charged tRNA from entering (like a bouncer)

Peptidyl-transferase

Forms a peptide in between the P and A site

What does the ribosome need for translocation?

GTP

Energy cost for Protein Synthesis

Charging of tRNA: 2 ATPs

Binding of tRNA to ribosome: 1 GTP

Translocation: 1 GTP

Total cost: 4 high energy phosphate bonds for each peptide bond formed

Termination

• The A site of the ribosome recognizes a STOP codon

• STOP codons (UGA, UAG, UAA) signal termination

• There are no tRNAs for any of the STOP codons

• Release factors bind to A site of ribosome

• Facilitates the release of the peptide chain and dissociation of the ribosome and mRNA