DIAGNOSTIC GENOMICS

Lecture 5
Expression of the
Genome - translation

Dr Shane Colley
College of Science, Health, Engineering and Education
Medical, Molecular and Forensic Sciences
Learning objectives
• Review Translation
• Roles of mRNA, tRNA and Ribosomes
• Initiation and importance of Kozak sites, elongation, termination, ribosome recycling
• Codons in mRNA, aminoacyl-tRNA synthases, elongation factors
• Redundancy in the genetic code, synonymous codons and effect of codon bias
• Proteins form three dimensional structures
• Primary, secondary, tertiary and quaternary structures
• Effects of point mutations on aa composition and protein structure/function
• Reading frame mutations
• Describe examples of Post-translational modification and targeting
• Processing of primary peptide
• Cleavage, chemical modification
• Localisation
• NLS, MLS, other organelles
• Signal transduction
• Examples of pathologies associated with altered translation
Translation
• Process by which Ribosomes synthesize polypeptides according the
code in an mRNA template
• mRNAs contain signals for ribosomal attachment, translational
initiation, amino acid (aa) sequence and peptide termination
• Range of elements required to get from mRNA codons to primary
amino acid sequence
mRNA codes for protein
• Information required to translate protein is encoded in mRNA transcribed
from genomic DNA
• Code is in 3 nucleotide (nt) triplet codons, with each codon specific for an
individual amino acid (aa)
• As the genome is a “4 base” code and codons are composed of 3 nts, there
are 64 codons (3nts/codon, 4 choices of nt/position results in 4 x 4 x 4 = 64)
• Distinct sequences in the mRNA specify translation start and stop sites with
intervening codons defining the linear sequence of the protein
• Codons code for either an aa or translation termination (ie a stop codon)
tRNA and the genetic code
• Two processes essential to translation
• 1) deciphering of triplet codons
• 2) correct incorporation of amino acids into peptides
• tRNA (transfer RNAs) link these two processes
• tRNAs
• 75-94 nts in length
• Each specific to a particular aa
• Contain an “anti-codon” that recognises/base pairs within the mRNA codon
• Enzyme responsible for attaching aas to tRNAs are aminoacyl-tRNA synthetases
• Translational fidelity comes from the correct aa being loaded into the tRNA and
interaction between the tRNA anti-codon and mRNA codon
• Fidelity is less important than for DNA replication as errors are non-inheritable
• Translation factors assist with translation
• Frequently GTPases (G proteins) that provide energy for ribosomal processes
Ribosomes
• Facilitates tRNA and mRNA interaction & peptide bond formation
between aas carried by tRNAs to yield proteins
• Composed of ~1/3rd protein and ~2/3rds RNA – ribosomal RNA (rRNA)
• 2 subunits
• Small mediates interactions between t and mRNAs
• Large catalyses peptide bonding between aas
Translating mRNA into protein
• Genetic information encoded within the mRNA is read by the
ribosome in the cytoplasm
• Transfer RNAs (tRNAs) act as a physical link between the coding
information and the pool of aas required to synthesise proteins
• tRNAs sequentially reads/decodes the codons in a 5’ to 3’ march
along the mRNA, recruiting aas to the growing protein in the order
specified by the mRNA.
• Incoming aas are chemically linked together during synthesis
• The ribosome is the structural scaffold facilitating the process
Translation steps
• Initiation
• Elongation
• Termination
• Ribosome recycling
Translation cycle - initiation
• Initiation requires AUG (also AUA in mitochondria) codon recognition
by the ribosome
• Ribosomes “scan” mRNA beginning at the ‘5 cap
• This defines the “open reading frame” (ORF) ie the 3 nt codon
sequence that codes for a protein & contains NO stop codons
• Initiation requires ribosomes, initiator methionine tRNA, and ~28
initiation factors
• Multiple ribosomes can occupy the same mRNA = polysome
Variation in codons for amino acids
• Only one codon for Met & Trp but 6 for Arg, Leu, Ser
Synonymous Codon Usage & Codon bias
• Synonymous codons code for the same aa
• Although coding for the same aa, different codon usage results in altered
translational efficiency - selective Bias towards certain codons.
• Not all tRNAs are equally abundant and some codons are used less frequently than others
• If silent mutations resulting in a change to a sub-optimal codon this can result in
reduced translation
• Argument that the inclusion of a rare codon may increase translational fidelity
due to greater pausing and checking
• May also influence RNA secondary structure, transcription factor binding, splicing
regulation

J Biol Chem. 2010 Sep 10;285(37):28741-8. doi: 10.1074/jbc.M110.154575. Epub 2010 Jul 13.
A synonymous single nucleotide polymorphism in DeltaF508 CFTR alters the secondary structure of the mRNA
and the expression of the mutant protein.
 Efficient initiation is Kozak site dependent
• mRNAs are generally monocistronic ie code for a single protein
• Initiation usually occurs at the first AUG encountered by the ribosome although
downstream AUGs may be favoured
• Efficient initiation is Kozak site dependent. Without site being intact, translation is
sub-optimal
• GCCGCC(A/G)(C/A)XAUGGCG

• Note: Nco 1 site = c/catgg frequently present in expression vectors

 Translation cycle - elongation
• Following initiation, aas are added to the initiating methionine of growing
polypeptide as the ribosome moves 5’ – 3’ along the mRNA
• Elongation factor eEF1A plays a role in loading aminoacyl-tRNAs into the ribosome
• Other EFs provide energy, prevent inappropriate interactions with tRNAs or other
translational elements, move the mRNA/tRNA complex through the ribosome
• Peptide bonds formed between correctly loaded/positioned aas within the
ribosomal complex
• Elongation continues until a stop codon is encountered at the end of the ORF
Translation termination and ribosome recycling
• At the end of an ORF the ribosome encounters an UAA, UAG or UGA
stop codon
• Translation is terminated and peptide is released from ribosome
• Stop codons recognised by class 1 release factors not tRNAs
• Ribosome dissociates into its small and large subunits
 Reading frame mutations
• There are no “gaps” in ORF
• Insertion/deletion (indels) will alter
reading frame
• As 1 in 20 codons = stop, alterations
will lead to premature termination
on average within 10 aas
• Point, splicing, inaccurate repair can
lead to frame shifts
 Point mutations
Mutations to individual nucleotides change codons and resultant peptide
sequence
 Amino acids have different biochemical properties
• AAs may be
• Polar, non-polar
• Aliphatic vs aromatic
• Negatively or positively charged
• Substrates for modification
• Serine, threonine, tyrosine
• Cysteine – thiol (s-s) bonding
• Substitutions between aas with similar
biochemical properties may “conserve” function
• Non-conserved changes may have marked
consequences
• Loss of structure/bonding – cysteine, proline
• Loss of cleavage site
• Changed localisation
Primary structure is only part of the story
Protein structure dependent on bonding
Intramolecular – within a protein
Intermolecular - between proteins

Covalent
(a)- disulfide bonds - cysteines

Noncovalent (weaker but diverse)

Do not share a pair of electrons
(b)- hydrogen bonds – aspartic acid + arginine
(c)- ionic bonds – between +ve and –ve aas
(d)- van der Waals interactions
(e)- hydrophobic interactions

Change aa sequence, change protein structure/function

Post translational modification
• Most proteins do not emerge from the ribosome in their fully
functional state
• They may be cleaved and/or post-translationally modified
• Folded into correct 3 dimensional shape - chaperones
• Clotting enzymes are synthesized in inactive form ready for cleavage &
activation; Chymotrypsin pancreatic enzyme
• Covalent addition of lipids – membrane localisation
• Phosphorylation – signal transduction
• Incorrect modification can lead to disease
Peptide localisation signals
 Mitochondrial localisation

Mitochondrial localisation signal – amino terminal positively charged amphiphilic alpha helixes
Amino-terminal mitochondrial signal “capped” with short peptide in lower panel – loss of mitochondrial localisation (green)
Case study demonstrating effect of non-
coding variation
• Case for illustration only
• Not expecting memorisation of disease, genes, variations involved in
this case
• Consider workflow & how might this point to genes involved
• phenotype
• Pedigree
• Investigation
• Identification of candidates
• validation
• Atrial septum defect (ASD) is a pathology involving the muscle walls of the heart
• Several transcription factors implicated including GATA4.
• GATA4 is a zinc finger TF that regulates the expression of genes including atrial
natriuretic factor (Nappa) and gap junction protein alpha 5 (Gja5)
• Multiple examples of GATA4 mutations being associated with ASD.
Case report
• Proband (individual first reporting with a heritable condition in a
pedigree) diagnosed with ASD and aberrant pulmonary vein draining
• Six spontaneous abortions prior to 12th week of gestation with no
identifiable cause
• Normal karyotype (microdeletion of 22q11 excluded as being causal)
• Mother had similar symptoms.
• No evidence of congenital heart defects in siblings or children.
Screening for cause
• Known candidate genes for ASD were assessed by sequencing
• A -6 G to C conversion in proband of GATA4 gene identified
• Position highly conserved for this gene between species and in other
Kozak sites
• Variant not identified in other data bases eg Exome Variant Server or
dsSNP
• Mother and siblings found to carry same variation
GATA4 promoter variant pedigree
Validation variant is associated with disease
• If translation is reduced as a result of the variation, expect lower
protein levels
• Biopsies of cardiac tissue….?
• Alternative….compared translational output of “reporter gene”
coupled to wt and variant promoter in vitro (in HeLa cells)
• Results
• Markedly lower wt protein expression with variant promoter.
• Evidence of a smaller product – leaky scanning resulting in use of downstream
AUG (read through of first AUG)
Less GATA4 protein from variant vs wild type transcript.
Consider
• Change to genome may not change a gene’s coding domain but can
cause the loss of protein expression
• Can involve
• Transcription initiation – loss of TF binding site
• Transcriptional stability – silent mutation altering mRNA stem-loop structure,
loss of PA signal
• Translational initiation - disturbed Kozak
• Protein synthesis – reduced translation with altered codon usage
• Protein stability/modification – changes in expression of protein modification
enzymes (change to another gene affecting gene your investigating!)
Exam question
• Describe the essential structural features of a 1,200 nucleotide long
mRNA that would allow it to efficiently translate a 200 amino acid
protein. A diagram may assist with the description.
• this question spans both lectures 4 &5
References
• Craig et al., 2014 Biology Principles of Genome Function 2nd Ed.
Chapters 1, 11, 14