Protein Synthesis

Key Terms

Translation - where a proteins is synthesised from an mRNA molecule at the ribosomes.

Proteins

Functions


Enzymatic - Catalyze biochemical reactions

  • Lactase enzyme breaking down lactose into glucose and galactose.


Regulatory - Involved in gene expression, such as proteins that act as repressors or activators


Structural - provides structural support

  • Collagen molecules forming collagen fibers


Transport - Facilitates the movement of ions and molecules across cell membranes

  • Channel and carrier proteins



Structure

  • Polypeptide chains are formed via peptide bonds, linking amino acids together.

  • The sequence of amino acids in a protein represents its primary structure.


  • Secondary Structure: Interaction with neighboring amino acids leads to folded structures such as alpha-helices and beta-pleated sheets.

  • Tertiary Structure: Interaction of secondary structures leads to the overall 3D conformation.

  • Quaternary Structure: Association of multiple polypeptide chains, potentially forming functional units called domains.


Amino Acids

Amino acids (aa) are the monomers of proteins. The general structure consists of:

  • Carboxyl group: COOH.

  • Amine group: NH2.

  • Variable group (R): Determines the properties of the amino acid

Different classifications of Amino Acids Side Chains

  • Electrically positively charged - Arginine (Arg), Histidine (His), Lysine (Lys).

  • Electrically negatively charged - Aspartic acid (Asp), Glutamic acid (Glu)

  • Polar Uncharged - Asparagine (Asn), Serine (Ser), Threonine (Thr), Glutamine (Gln), Cysteine (Cys), Proline (Pro).

  • Hydrophobic - Alanine (Ala), Valine (Val), Leucine (Leu), Isoleucine (Ile), Methionine (Met), Phenylalanine (Phe), Tyrosine (Tyr), Tryptophan (Trp).


Non-essential amino acids: Can be synthesized by the body (e.g., Alanine, Serine, Proline).

Essential amino acids: Must be obtained from the diet (e.g., Isoleucine, Methionine, Phenylalanine).


Codons

Codon - A triplet of 3 nucleotides that code for 1 amino acid.

Total Codons: 64 possible codons are identified:

3 Stop Codons: UAA, UAG, UGA.

61 Sense(normal) Codons: Each coding for specific amino acids.


The genetic code is described as:

  • Degenerate: More than one codon may specify a particular amino acid.

  • Not Ambiguous: No codon specifies more than one amino acid.

  • Reading Frame: Codons must be read sequentially without overlap, starting from a designated initiation codon (AUG).

  • Universal: Genes can be transcribed and translated after being transferred from one species to another


RNA

Three main classes involved in translating genetic information:

  • Messenger RNA (mRNA): Carries genetic information from the nucleus to the ribosome.

  • Ribosomal RNA (rRNA): Component of ribosomes, facilitating protein synthesis.

  • Transfer RNA (tRNA): Links amino acids to codons on the mRNA during translation.


tRNA

  • tRNA functions as the bridge between the genetic code and the amino acids in proteins.

  • Each tRNA carries a specific amino acid and binds to the correct codon on the mRNA through its anticodon.


Structure

  • 74 - 95 nucleotides long

  • CCA sequence at the 3’ end

  • Anticodon three-base sequence complementary to the mRNA codon.

  • D-loop and T-arm assist enzyme recognition and ribosome binding


D-loop - Contains the unusual base dihydrouridine (D).

  • Important for tRNA recognition by aminoacyl-tRNA synthetase, the enzyme that attaches the correct amino acid.


T-arm - Contains the sequence ribothymidine (T), pseudouridine (Ψ), and cytidine (C).

  • Helps the tRNA bind to the ribosome during protein synthesis.

rRNA

ribosomal RNA, a non‑coding RNA transcribed by RNA polymerases (Pol I in eukaryotes, a single RNA Pol in prokaryotes).


  • rRNA + ribosomal proteins = ribosome, but rRNA is the dominant component (≈60% of ribosome mass).


Structure

  • 60S large subunit - (28S, 18S, 5.8S)

  • 40S small subunit - (5S)


Translation

What: mRNA is translated into proteins.

When: Translation occurs after mRNA is transported from the nucleus to the cytoplasm.

Where: Ribosomes, starting near the 5' end of mRNA.


Stages

  1. tRNA Charging: Binding of tRNAs to their corresponding amino acids.

  2. Initiation: Assembly of all necessary components at the ribosome.

  3. Elongation: Addition of amino acids to the growing polypeptide chain.

  4. Termination: Completion of protein synthesis at stop codons.


tRNA charging

  • The CCA sequence is present in all tRNAs, with amino acids attached to the 3' end.

  • Specificity determined by aminoacyl-tRNA synthetases based on nucleotide sequences and properties of amino acids.

  • recognition of tRNA by the aminoacyl-tRNA synthesis is mediated by the nucleotide sequence

  • Recognition of amino acid by the aminoacyl-tRNAsynthesis is mediated by size, charge and R groups


Aminoacyl-tRNA synthesis (or aminoacylation) - attaching an amino acid to its correct tRNA.

Initiation

What is needed

  • mRNA

  • small and large subunits of the ribosome

  • initiation factors

  • initiator tRNA (met-tRNA)

  • GTP


Stages

  1. Binding of Components: mRNA, ribosomal subunits, initiation factors, and initiator tRNA.

  2. Pre-initiation Complex (43S): Recognizes the 5' cap on mRNA and scans for AUG codon.

  3. Kozak Sequence: Surrounding sequence that aids in recognizing the initiation codon.


Basic

  1. mRNA binds the small subunit of the ribosome

  2. Initiator tRNA binds to the mRNA (anti-codon binding)

  3. The large ribosomal subunit joins the complex


Detailed

  • the small (SSU) and large (LSU) ribosomal subunits need to be separate for the mRNA to bind the small subunit

  • a 43s Pre-initiation complex composed of SSU, met-tRNA a and initiation factors recognise and bind the 5’cap in the mRNA

  • 43s pre-initiation complex scans the mRNA until the 1st AUG codon is found

  • AUG is surrounded by a consensus sequence which helps the recognition of the Kozak Sequence (ACCAUGG)

  • After recognition, codon and anticodon bind

  • initiation factors are released

  • LSU binds to the complex

  • CBC (cap-binding complex) promotes the nucleus to cytoplasm export and checks for errors

  • CBC is replaced by ELF-4E for continuation

  • poly-A interacts with the 5’cap via regulatory proteins and formation of a closed loop. promoting stabilisation of SSU/mRNA binding


Initiation of translation

  • 12 IFs are needed which mediate

    • preventing binding of LSU via binding to SSU

    • recognition and binding of the 5’cap

    • recruitment of the initiator tRNA

    • binding between the initiator tRNA and the initiator codon

    • LSU binding


Key molecules

43s Pre-initiation complex - Group of molecules the come together before translation begins.


Includes

  • small ribosomal subunit (40s)

  • initiation factors ( eIF1 ,2 , 3 )

  • initiator tRNA carrying methionine

  • GTP -for energy


Met-tRNAiMet (methionyl initiator tRNA) - tRNA molecule that carriers the 1st amino acid methionine which starts protein synthesis.

  • i - initiator so only used fro starting

  • Met-tRNAiMet pairs with the start codon AUG


Consensus sequence - pattern of bases found in DNA / RNA that helps proteins know where to bind or start a process


EIF - eukaryotic initiation factors hep start protein synthesis


Elongation

Involves binding of charged tRNA to A site, formation of peptide bonds mediated by rRNA, and movement of ribosome (translocation).


Requires

  • 80s initiation complex

  • charged tRNA

  • elongation factors

  • GTP


3 possible binding sites for tRNA

  • Aminoacyl (A)

  • Peptidyl (P) - occupied by initiator

  • Exit (E)


3 steps

  • Binding

  • Formation

  • Translocation


In Detail


Binding

  1. a charged tRNA will bind to site A, this is helped by EIF eEF1 alpha, which is bound to GTP

  2. anticodon on tRNA pairs with the complementary codon on mRNA

  3. GTP is hydrolysed to GDP, and eEF1 alpha is released from the ribosome.

  4. other eEFs then convert GPT back to GTP so eEF1 can be reused

  5. amino acid in A site joins to the growing polypeptide chain attached to tRNA in the P site


Formation

  1. peptide bond forms

  2. causing the growing polypeptide chain to be transferred in the a Site

  3. tRNA in p site loses its amino acid and become uncharged

  4. this is in the LSU and is catalysed by 28s rRNA (ribozyme


Translocation

  1. ribosome moves one codon along the mRNA in the 5’ to 3’ direction

  2. it is powered by eEF2 and GTP hydrolysis

  3. tRNA stays attached to the mRNA while ribosome moves

  4. the tRNA carrying the growing polypeptide moves from the A site to the P site.

  5. the empty (

  6. uncharged) tRNA moves from the P site to the E (exit) site.

  7. the empty tRNA leaves the ribosome and enters the cytoplasm, where it can be recharged with another amino acid.



Termination

Process


The ribosome reaches a stop codon

  • Translation ends when the ribosome reaches a stop codon on the mRNA (UAA, UAG or UGA).

  • There is no tRNA with an anticodon that matches a stop codon, so the A site remains empty.

  • Instead of a tRNA entering the A site, release factors (RFs) bind to the ribosome.


eRF1 recognises the stop codon

  • eRF1 (eukaryotic Release Factor 1) enters the A site.

  • eRF1 recognises and binds to the stop codon.

  • This signals that translation should end.


eRF3 releases the completed polypeptide

  • eRF3 works together with eRF1.

  • eRF3 uses GTP hydrolysis to provide the energy needed for termination.

  • This causes the bond between the tRNA and the completed polypeptide (tRNA–polypeptide bond) in the P site to be cleaved (broken).

  • The completed polypeptide (protein) is released from the ribosome.


The ribosome is recycled

  • Other release factors (RFs) help:

    • release the empty tRNA,

    • release the mRNA, and

    • separate (dissociate) the large and small ribosomal subunits.

  • The ribosomal subunits, mRNA and tRNA can now be reused in another round of protein synthesis.