Translation: Protein Synthesis

Translation (Protein Synthesis)

• DNA in the nucleus acts as a template
• mRNA is processed before exiting the nucleus

mRNA Information

• When mRNA is formed, it contains codons
• mRNA goes into the cytoplasm and attaches to ribosomes

tRNA actions

• tRNA, possessing an anticodon, transports specific amino acids to mRNA
• tRNA will leave and soon carry another amino acid
• Anticodon-codon complementary base pairing takes place

Protein Creation

• A peptide chain is created from tRNAs
• The peptide moves from resident tRNA to incoming tRNA

Ribosome Components

• The ribosome is where the protein synthesis takes place
• The ribosome is a protein comprised of two subunits that vary in size
• mRNA is read by the ribosome in a 5' to 3' direction

Ribosome Structure

• Three tRNA binding sites are present

tRNA Binding Sites

• Exit (E): spent tRNA depart from here
• Peptidyl (P): The tRNA bound to the growing oligopeptide connects to this site
• Aminoacyl (A): The tRNA, bound to an amino acid, attaches to this site

Central Dogma

• One gene will translate into one protein
• The central dogma of molecular biology describes how genetic information moves from DNA to RNA to generate a protein that performs a function

DNA Information

• DNA contains information needed to create proteins
• RNA carries the information from DNA to the ribosome

Genetic Information Order

• Genotype (DNA) -> transcription -> translation -> protein

Protein Synthesis Definition

• The process of using the genetic code to make proteins is called protein synthesis, and it has two steps: transcription and translation

Transcription

• Occurs when the coding part of a gene is copied to RNA

Translation

• The manufacturing of proteins from mRNA

Basic Principles

• Transcription is RNA synthesis from DNA
• Transcription yields messenger RNA (mRNA), which is a transcript
• Translation involves polypeptide synthesis utilizing mRNA
• Ribosomes perform translation activities

Translation and transcription in Prokaryotes vs Eukaryotes

• In prokaryotes, mRNA translation can start before transcription is done
• In Eukaryotes, the nuclear envelope separates transcription from translation

Types of RNA

• There are three types of RNA, messenger RNA (mRNA), transfer RNA (tRNA), and Ribosomal RNA (rRNA)

Messenger RNA (mRNA)

• mRNAs are the genetic coding templates utilized by translation to establish the order of amino acids for a polypeptide

Transfer RNA (tRNA)

• Tiny RNAs that covalently affix to particular amino acids, identifying mRNA sequences to appropriately insert amino acids in the polypeptide chain

Ribosomal RNA (rRNA)

• Assembled with ribosomal proteins to create ribosomes
• Ribosomes interact with mRNAs to generate the catalytic domain for tRNA to enter to allow proteins to catalyze

Codons and Amino Acids

• Each codon is specific to the amino acid that will be added to the corresponding position along a polypeptide

Genetic Code Note

• The genetic code is redundant (an amino acid is specified by multiple codons) , but it is not ambiguous
• An amino acid cannot be specified by more than one codon

Protein Biosynthesis

• Involves 5 stages: activation, initiation, elongation, termination and folding/ post-translational processing

Protein Biosynthesis - Activation of Amino Acids Requirements

• Two chemical requirements are to activate the carboxyl group of each amino acid to allow creation of a peptide bond and to create a link between each new amino acid and mRNA data

Amino Acid Activation

• Proper amino acid attachment to the right tRNA is extremely important
• Takes place in the cytosol, not ribosome
• Each amino acid covalently attaches to a specific tRNA using ATP and Mg²-
• Aminoacyl tRNA synthetases are the activating enzymes for each reaction
• Amino acid + tRNA + ATP + Mg2+ -> aminoacyl-tRNA + AMP + PPi
• tRNAs are referred to as "charged" when attached to amino acids via aminoacylation

Protein Synthesis - Initiation Requirements

• This process in prokaryotes needs the start codon AUG (methionine) in the presence of initiating factors in the protein structure (IF 1, IF 2 and IF 3), ribosome 30S unit, MRNA, N-formyl methionyl-tRNA, and GTP (Guanosine triphosphate)

Prokaryote Protein Synthesis - Initiation Factors

• The process involves initiating factors (IF) and GTP to form the ribosome subunits, and mRNA and fMet-tRNA in close proximity to the mRNA’s 5' end
• A site on the initiation complex links peptidyl-tRNA to the P-site and connects aminoacyl-tRNA
• The fMet-tRNA and another mRNA codon are in situ in the initiation complex P and A complex, respectively.

Initiation Process - Step 2

• A complex made up of the 30S ribosomal subunit, IF-3 and mRNA is linked by IF-2 and initiating fMet-tRNAfMet with GTP
• The mRNA connects and the initiator codon is placed in the right spot via the Shine Delgarno sequence
• The anti-codon of this tRNA now in alignment with the mRNA initiation codon

Shine-Dalgarno Sequence

• The Shine-Dalgarno sequence is a ribosome binding site, required for translation intiation
• The ribosome doesn't bind to the AUG, instead to 5-10 nucleotides upstream and can be anywhere within an mRNA

Initiation Stage - Step 3

• In Step 3 the complex merges with a 50 ribosomal subunit where GTP bound to IF-2 is hydrolyzed to Pi and GDP and secreted from the complex alongside three initiation factors

Eukaryotic Initiation

• Translation is nearly the same in both eukaryotic and bacterial cells, with initiation mechanics being a primary distinction only
• Eukaryotic mRNAs attach as a complex to the ribosome via specific binding protein
• Several proteins join the 5' and 3' ends of the message

Poly(A) Binding Protein (PAB)

• At the 3' end of mRNA, it is bound by Poly(A) Binding Protein

Eukaryotic Cells

• A minimum of nine initiation factors exist: forming a complex called eIF4F, which contains eIF4E, eIF4G and eIF4A, and attaches to the 5' cap by means of eIF4E
• The protein eIF4G binds to eIF4E and PAB proteins to tie the complex together
• The eIF4F is the protein for the eIF4 complex that connects to the RNA molecule

Polypeptide Chain Elongation

• An aminoacyl-tRNA interacts with the binding site of the ribosome
• A polypeptide chain in development is then moved from the tRNA in the P-site to the new tRNA in the A-site by building new peptide bonds
• The ribosome moves and positions the mRNA’s next codon into the A binding site causing nascent polypeptide-tRNA to translocate from A to P, and uncharged tRNA from P to the E site

Termination and Release

• Elongation proceeds until MRNA’s last amino acid is coded, then it terminates following the “stop” codon: UAA, UAG, or UGA
• Termination factors (or release) will dissociate the ribosomal complex after they interact with A

Termination Requirements

• A water molecule will then bond near the nascent polypeptide’s carboxyl terminus, completing the process when a terminal peptidyl-tRNA bond is disolved
• As the subunits loosen, any free polypeptide as well as the last, uncharged tRNA will remove from the P site

Folding and Posttranslational Modifications

• Peptide chains are connected ("folded") to form globular proteins by forming disulfide bonds and attaching cofactors/co-enzymes in addition to prosthetic groupings
• Peptides are changed for use in chemical activities
• An example of this process is the addition of cabohydrates makes a glycoprotein
• Precursor proteins are used as well. For example, proinsulin -> insulin
• There are also chemical modifications such as, methylation and acetylation