Topic E - Translation

Amino Acid

Each amino acid contains a central carbon bonded to; An amino group (NH3+), a hydrogen, a carboxyl group (COO), and an “R” (radical) group

-20 different amino acids, differ by chemical composition of R groups

1. Polar - able to interact with water (hydrophilic)

2. Nonpolar - unable to interact with water (hydrophobic)

3. Charged (acidic or basic)

-Adjacent amino acids are connected via peptide bonds, covalent bond between NH3+/COO

-Multiple amino acids joined to form a polypeptide chain

-Polypeptide chains have polarity:

1. Free amino group at one end (N terminus)

2. Free carboxyl group at one end (C terminus)

• Polypeptide chains are synthesized N terminus → C terminus

Protein Structure and Folding

Primary Structure - amino acid sequence, affects all other structures

Secondary Structure - formed through hydrogen bonds between COO and NH3+ groups in polypeptide backbone

1. Alpha Helix

2. Beta Strand (can then form Beta sheets)

3. Unstructured loops

Tertiary Structure - interactions between side chains

Quarternary Structure - multiple 3D polypeptide chains

Gene, mRNA and Polypeptide Structure

Gene Structure

-Regulatory region (promoter)

-Transcribed region

-Translated region

mRNA

-Transcription start site

-Transcription termination site

-5’ and 3’ UTR

Polypeptide

-Translation start codon: AUG

-Translation stop codon: UAG/UAA/UGA

Codons and Reading Frame

Codons: Set of three nucleotides

-61 “sense” codons and 3 stop codons

-Contains degenerate codons (codons that code for the same amino acid)

Reading frame is non-overlapping (one nucleotide is only part of a single codon)

-The correct reading frame will contain the “start codon” (AUG)

Components of Translation

mRNA - carries information to make a protein

Amino Acid - building blocks of a protein

Ribosome - enzyme that catalyzes the formation of peptide bonds

-made up of rRNA and proteins

-Small subunit and large subunit

-A, P, and E sites

tRNA - deliver amino acids to ribosomes

tRNA structure

Amino Acid Attachment Site

-tRNAs can be charged (bound to amino acid) or uncharged (not bound to an amino acid)

-Amino acids are attached to tRNA via aminoacyl tRNA synthetase

-Different tRNA for each amino acid

Anticodon

-Interacts with codon in mRNA by complementary base pairing

-5’ end of anticodon can be promiscuous (doesn’t require strict base pairing)

• 5’ end has weak base pairing with 3’ end of codon in mRNA, called “wobble base”

• Explains the redundancy of genetic code

Stages of Translation

Initiation: Ribosome and initiator tRNA bind to mRNA

Elongation: Ribosome synthesizes polypeptide

Termination: Ribosome stops synthesizing polypeptide. Translation complex disassociates and polypeptide is released

Translation Initiation in Bacteria

The ribosome binding site is a specific sequence in the 5’ UTR of the mRNA (also called “shine-dalgarno sequence”)

-Shine-dalgarno sequence is recognized by complementary base pairing between rRNA of the small subunit and the mRNA

-Positions the P site directly on top of the AUG start codon

-Complementary tRNA binds in the P site

-Ribosome large subunit will then bind, beginning translation

Translation Initiation in Eukaryotes

Small subunit and initiator tRNA bind to the 5’ cap

-Complex scans the mRNA for a start codon

-Indicated by the Kozak sequence (sequence that surrounds the start codon)

-Large subunit binds allowing for translation

Translation Elongation

Decoding:

A site - charged tRNA binds to complementary codon containing the next amino acid to be added

Peptide Bond Formation:

P site - peptide bond formation between the amino acid attached to tRNA in the A site and the polypeptide chain attached to tRNA in the P site

-Polypeptide is transferred to tRNA in the A site from the tRNA in the P site

Translocation

E site - Ribosome moves down (towards the 3’ end of the mRNA), uncharged tRNA moves to the E site and leaves the ribosome

-tRNA in A site → P site, tRNA in P site → E site

mRNA is translated by multiple ribosomes at the same time

Translation Termination

Stop codon in A site of the ribosome

Release factor binds to A site of the ribosome

-Causes hydrolysis of polypeptide chain from P site tRNA

-Polypeptide chain is then released

Ribosome, mRNA and tRNAs dissociate

Antibody

Protein that recognizes and binds to specific region (epitope) on a specific protein (antigen)

-Consists of a variable region (different binding site for different antigens) and constant region (same amino-acid sequences)

-Used by the immune system

Can be labelled in order to be visualized

-Fluorescence

-Enzymes that produce light or color

Made by injecting a lab animal with a protein of interest, isolate antibodies from the blood

Primary and Secondary Antibodies

Primary Antibody - binds to the protein of interest

Secondary Antibody - binds to the primary antibody and is labelled

-Primary antibodies can be difficult and expensive to obtain, while secondary antibodies can be mass produced easier

SDS-Page Technique

SDS denatures proteins and coats them in a uniform negative charge

-Also uses heat to denature proteins before electrophoresis

-Sorts polypeptides according to size (smaller proteins at the bottom, larger protein at the top)

-All proteins can be visualized using a non-specific stain (Coomassie is the most common, but also silver staining and ponceau)

Western/Immunoblotting

Used to visualize a specific protein

-Run SDS page

-Transfer proteins from a gel to a membrane/filter paper via lateral electrophoresis

-Incubate with specific primary antibody, wash

-Incubate with labelled secondary antibody, wash

-Detect

Common Controls:

-Wildtype/healthy variant cells

-Loading control

Advantage and Disadvantages for Immunoblotting

Advantage

-Can look at gene expression

-Can determine if protein is present or absent from sample

-Semiquantitative (can compare relative abundances)

-Relatively simple technique

Disadvantage

-Not quantitative

-Limited sensitivity

Immunofluorescence

uses antibodies to determine the location of a protein of interest

-uses fixation and permeabilization to prevent cell function/open cells to allow for incubation with primary and then secondary antibodies (labelled with fluorescent molecules)