The birth and death of proteins

Some key concepts

 Proteins (polypeptides) are synthesised by translating the sequence encoded in the mRNA in the form of
a non-overlapping, degenerate triplet code.
 This triplet code dictates translation of specific RNA triplet codons to amino acids and occurs in the
cytosol.
 The AUG start codon for methionine is most common, specifying the NH2-terminus of most proteins
 An open reading frame (ORF) is the uninterrupted triplet sequence in mRNA from a specific start codon
to the stop codon, which is translated into a linear sequence of amino acids in a polypeptide chain.
 Mutations in the base sequence can alter ORF and subsequent proteins
Types of mutations
Deletions or Insertions: 1bp to several Mbp
Single base substitutions
Missense mutations: replace one amino acid codon with another
Nonsense mutations: replace amino acid codon with stop codon
Splice site mutations: create or remove exon-intron boundaries
Frameshift mutations: alter the ORF due to base substitutions
Dynamic mutations: changes in the length of tandem repeat elements

Translation requires 3 main components to come together

1) Messenger RNA:This class of RNAs are the genetic coding templates used by the translational machinery
to determine the order of amino acids incorporated into an elongating polypeptide in the process of
translation.
2) Transfer RNA: A class of small RNAs that form covalent bonds to amino acids and allows correct
insertion of amino acids into the elongating polypeptide chain.
3) Ribosomes: Ribosomal RNA (rRNA) assembled, together with numerous ribosomal proteins, to form the
ribosomes. Ribosomes engage the mRNAs and form a catalytic domain into which the tRNAs enter with
their attached amino acids. The proteins of the ribosomes catalyze all of the functions of polypeptide
synthesis

Translation has 2 important recognition steps

1) Correct aminoacylation (‘charging’): tRNA has to be charged (aminoacylated with the correct amino
acid) to allow the amino acid to participate in peptide bond formation. This is brought about by the
enzyme, aminoacyl-tRNA synthetases (aaRSs) which covalently attach the correct amino acid to
tRNA (specified by anticodon) in an ATP dependent process. The specificity of aaRSs to select the
right tRNA to be acylated (especially since most tRNAs are structurally similar) is done by
recognising specific tRNA identifiers present on the acceptor step & anticodon loop. e.g. AlaRSs
recognise G3.U70 bp.
2) Select the correct charged tRNA as specified by mRNA: Less than 61tRNAs are found in cells (based on
conventional Watson-Crick base paring), which suggests that a single tRNA anticodon would
recognise more than one mRNA codon. This is the ‘Wobble hypothesis’ which explains this non-
standard pairing of the 3 base position of the mRNA (codon) with the 1 st position on the tRNA
(anticodon).
Polypeptide synthesis (translation) can be divided into 3 main steps
1) Chain Initiation
Initiation usually begins at the first AUG from the 5’ end of the mRNA. Recognition of the codon is
mediated by a series of eukaryotic initiation factors (eIFs). The ribosome assembles along with the
mRNA and charged initiator tRNA (tRNAimet). The small (40S) and large subunit (60S) are usually
kept apart by eIF3 and eIF6 when not engaged in translation. Initiation is a 4 step process as follows
Step 1: Formation of pre-initiation complex. The 40S-eIF3 is bound by eIF1A to a ternary complex of
tRNAimet, eIF2 and GTP
Step 2: Formation of initiation complex (cap binding of mRNA to 40S)
Step 3: Positioning at start codon. The initiation complex slides along mRNA using the helicase activity
of eIF4. eIF4 uses ATP hydrolysis to unwinds the mRNA secondary structure. Initiation complex
stops at the start site AUG. This recognition allows an irreversible GTP hydrolysis of eIF2
preventing any further unwinding
Step 4: Association of large subunit (60S). Irreversible GTP hydrolysis mediates the association of 60S-
eIF6 (large subunit ) to the small subunit by the action of eIF5. This becomes the P site
2) Chain Elongation
This step is mediated by eukaryotic elongation factors (eEFs). The main steps in this process involve
entry of the aatRNA, conformational change in the ribosome structure, formation of the peptide bind
and the translocation of the ribosome to the next codon on the mRNA. These can be summarised as
follows
Step 1. aatRNA binding
aatRNA binds to A site on ribosome by base pairing with codon
Step 2. Conformation change in ribosome: induced by GTP hydrolysis of EF1a
Step 3: Transpeptidation
C terminal of polypeptide uncoupled from P site tRNA and peptide bond transferred to amino acid
on A site tRNA
catalysed by peptidyltransferase
Step 4: Translocation
GTP hydrolysis of EF2 causes 2nd conformational change P site tRNA is transferred to E site
Simultaneous transfer of A site tRNA moved to P site
3) Chain termination
Release factors recognise and bind to stop codons. This induces peptidyl transferase to transfer peptidyl
group to water instead of aatRNA and the nascent polypeptide is released. Uncharged tRNA is released
from the 80S ribosome and the inactive ribosome then releases the mRNA.
Post translational folding and / or modification
The amino acid sequence of a protein determines its folding into a specific 3-D conformation. This folding
is mediated by molecular chaperones (e.g. Hsp70) or chaperonins (Hsp60 complexes). Nearly every
protein is modified after synthesis on the ribosome. These modifications are essential and dictate the
activity, life span or the cellular location of proteins. During modification, various chemical groups (e.g
acetyl, phosphoryl, hydroxyl, glycosyl etc) are added to the N or C terminal or within internal residues
of the polypeptide.
Death of proteins: Proteins that are misfolded, denatured, in excess or extracellular in origin are targeted
for degradation within lysosomes. Another pathway is by the addition of ubiquitin to lysine residues,
which is recognised are destroyed by the proteosome complex. Degradation of proteins can be a part of
normal cell processes (cell cycle) or may be implicated in disease, especially neurodegenerative diseases
(Parkinsons. Alzheimers etc).