Download Deciphering the genetic code Dr. Syndey Brenner estabilished mRNA


Deciphering the genetic code
o Dr. Syndey Brenner
 estabilished mRNA as intermediate
 Discovered triplet nature of code
 Developed C. elegans as molecular model
o Marshall Nirenburg
 Polyurdylic aci translation
 Codons associated with specific AA sequence

The genetic code
o Codon: 3 nudleotide base sequence
o Use the bases A, G, C, and U with 64 possible combinations
o Written 5’ to 3’
o Characteristics
 Specificity –unambiguous because a particular codon always codes for the
same amino acid

Universality – The specificity has been conserved from the early stages of
evolution (only small differences) in how the code is translated

Degeneracy –(or redundantcy) Each codon corresponds to a single amino
acid, but a given amino acid may have more than one triplet coding for it.
As a example, arginine is specified by six different codons. Methionine
and tryptophan have just one codon.

The wobble hypothesis:
o The first two bases of a codon always form strong
Watson Crick base-pair with the corresponding bases of
anticodon and confer most of the coding specificity.
o
The first base of anticodon [5 ’to 3’ direction] or the 3rd
base [3’ to 5’ direction] called the wobble base allows the
single t-RNA to adopt more than one codon. The 3rd base
[3’ to 5’ direction] of the codon leading to loose base
pairing which is termed “wobble”. The wobble permits tRNA to read more than one codon with the maximum
limits of three codons.
o
When the first base in the 5’-end in the anticodon is “U”
then the third base in codon can be either “A” or “G”. The
“U” forms strong Watson-Crick base pair with “A” and
wobble pairing with “G”. Similarly if “G” present at 5’
ends of anticodon, then “G” forms Watson-Crick base pair
with “C” and wobble base pair with “U”.
o When “I” or some other modified base present at 5’-end of
anticodon then t-RNA can recognize three different codons,
all of which form a wobble base pairing at 3’ position of
codon. The bases that can pair in this case are “A”, “U”
and “C”.

Non-overlapping and comma less – The code is read from a fixed
starting point as a continuous sequence of bases taken three at a time ACGUGGAUACAU is read as AUG-UGG-AUA-CAU with no breaks or
punctuation between the codons.
o Important codons
 Start: AUG (sets the reading frame)
 Stop: UAG, UGA, UAA
o Mutations
 Point mutation- a single base change
 Silent mutation – The codon containing the changed base codes
for the same amino acid. Example, if the serine codon UCA is
given a different third base – U – to become UCU it still codes for
serine


Missense mutations – The codon containing the changed base
may code for a different amino acid. For example, if the serine
codon UCA is given a different first base – C – to become CCA, it
will now code for proline or an incorrect amino acid (missense)
o Sickle cell anemia
 A point mutation in 6th codon of gene changes a
GAG  GTG
 During translation substitutes valine for normal
glutamic acid
 Causes cells to be sickle shaped
 Hereditary blood disorder with co-dominant

Nonsense mutation – The codon containing the changed base may
become a termination codon. For example, if the serine codon
UCA is given a different second base – A – to become UAA, the
new codon causes termination of translation (a nonsense mutation)
Frame-shift mutations – can change the reading frame if not 3 bases
 Insertion- an addition of one or more bases
 Deletion- a loss of one or more bases


Trinucleotide repeat expansion- a 3 base repletion in tandom
 Huntington’s
o CAG trinucleotide repeats which leads to defective protein
that cause neurodegeneration
 Fragile X syndrome
o Trinucleotde expanision in untranlated region causes
decrease in overall amount of protein so neurons degernate
during early development
o Also seen I nmyotonic dystrophy
Translation
o mRNA, ribosome, charged tRNAs, accessory factors, GTP for energy
o charging of the tRNA

aminoacyl-tRNA synthetase required for attachement of AA to
corresponding tRNAs
 two step reaction leading to the covalent attachment of the carboxy
group of the AA to the 3’ end of the tRNA
 reaction requires ATP
 proof-reading and editing activity
o the ribosome
 small ribosomal unit: bind mRNA and is responsible for the accuracy of
translation by facilitating correct base pairing between codon and mRNA
 large subunit: catalyzes formation of peptide bonds that link the AA in
growing polypeptide chain

3 sites of interaction between ribosome, tRNA, and mRNA
 A site: aminoacyl site where incoming charged tRNA attaches
 P site: the peptidyl site where the peptide bond is formed
 E site: exit site depleted tRNA is released
o Prokaryotes
 Can have multiple ribosome biding sites: Shine-Dalgarno sequences
 Recognition is facilitated by IF-2-GTP in prokaryotes and eIF-2-GTP
(plus additional eIFs) in eukaryotes
 In bacteria and in mitochondria the initiator tRNA carries an N-formylated
methionine
 GTP is hydrolyzed and 50S subunit is recruited with loss of IF’s.
 Note – in prokaryotes three initiation factors are known (IF-1, IF-2,
IF-3) whereas eukaryotes have over 10 initiation factors (eIF)
 The large ribosomal subunit then joins the complex, and a
functional ribosome is formed with the charged initiating tRNA in
the P site, and the A site is empty
 Formation of the peptide bond is catalyzed by peptidyltransferase, an
activity intrinsic to the 23S rRNA found in the 50S subunit
 The ribosome then advances three nucleotides toward the 3’-end of the
mRNA.
 This process is known as translocation and in prokaryotes requires
participation of EF-G-GTP and GTP hydrolysis (eukaryotes cells
use EF-2-GTP)
 Attenuation
 Excess tryptohphan: transcription terminated
 Tryptophan starved: transcription not terminated
 Transcription and translation can be coupled in bacterial cells
 Leader sequence
o POL
o Eukaryotes
 process is more complex compared to prokaryotes.
 also a site for regulation of protein synthesis
 Initiation of translation involves formation of a complex composed of :
 methionyl-tRNAi met
 mRNA
 a ribosome

Initiation:
 methionyl-tRNAi met initially forms a complex with the protein
eukaryotic initiation factor 2 (eIF2) which binds GTP (This
complex binds to the small (40S) ribosomal subunit)
 The cap at the 5’-end of the mRNA binds an initiation factor
known as the cap-binding protein (CBP or eIF4F)---CBP contains
a number of small subunits, including eIF4E
 Several other eIFs join, and the mRNA then binds to the eIFs-MettRNAiMet-40S ribosomal complex
 In this reaction, hydrolysis of ATP is required because a helicase
is needed to unwind the hairpin loop in the mRNA and scans the
mRNA until it locates the AUG start codon
 GTP is hydrolyzed, the initiation factors are released, and the large
ribosomal (60S) subunit binds (The ribosome is now complete).
o The 80S eukaryotic ribosome contains one small (40S) and
one large (60S) subunit and it has two binding sites for
tRNA, known as the P and A sites.

Elongation
 When Met-tRNA is bound to the P site, the mRNA codon in the A
site determines which aminoacyl –tRNA will bind to that site
 In eukaryotes, the incoming aminoacyl-tRNA first combines with
elongation factor EF1a containing bound GTP before binding to
the mRNA ribosome complex (EF1 is the GTP-binding a-subunit
of a heterotrimeric G-protein which is activated for association
with other proteins when it contains GTP)
 When the aminoacyl-tRNA –EF1-GTP complex binds to the A
site, GTP is hydrolyzed to GDP.
 This releases EF1-GDP from the aminoacyl –tRNA ribosomal
complex; proteins synthesis continues
 Free EF1-GDP re-associates with the EF1-subunits, and GDP
is released
 GTP binds and the bg-subunit dissociates.
 Then, EF1-GTP is ready to bind another aminoacyl –tRNA
 Contrast: Elongation in prokaryotes is similar – except that the
corresponding factor for EF1 is named EF-Tu and the associating
elongation factors are called EF-Ts instead of EF1.

Termination at the stop codon

Hormone regulation of protein synthesis
 Insulin (hormone) can regulate protein translation:
o Stimulates via activation of eIF4E (initiation factor)
o Normally, eIF4E is bound to an inhibitor protein, 4EBinding factor
o When insulin binds to it’s receptor that results in
phosphorylation of 4EBP and that releases eIF4E for
initiation of protein synthesis

Polyribosome (polysomes)
 Where multiple copies of polypeptides can be made at once

Post-translational modifications
 Various enzymatic modifications can occur to affect protein
function or structure
 Proteolytic cleavage removes the terminal 5’ Met, and other
proteases may cleave the protein.


Nine amino acid modifications
o Acetylation
o Methylation
o Hydroxylation
o Carboxylation
o Fatty acylation
o Prenylation
o ADP ribosylation
o Phosphorylation/dephosphorylation
o Glycosylation
 N-glycosylation occurs in the endoplasmic
reticulum and O-glycosylation in the Golgi

Proteins destined for secretion
o Initially functionally inactive
o Cleavage can occur or placed in secretory vesicles
o Zymogens are inactive precursors of secreted proteins and
become activated through cleavage after reaching a site of
action (example: pancreatic zymogen, trypsinogen,
activated to trypsin in the small intestine.
Chaperones- mediate posttranslational folding
 Foldases- support folding of proteins in ATP-dependent manner
 Holdases- bind folding intermediates to prevent aggregation

Diptheria toxin mechanism of action
 Diphtheria toxin is a bacterial AB toxin whose gene is encoded by
a virus that infects Corynebacterium diphtheriae.
 Diphtheria toxin B-subunit binds to a cell surface receptor,
facilitating the entry of the A-subunit into the cell.
 The A-subunit catalyzes a reaction where the ADP-ribose (ADPR)
portion of NAD is transferred to EF2 (ADP-ribosylation). ADPR is
attached covalently to a post-translationally modified histidine
residue, known as diphthamide.
 ADP-ribosylation of EF2 inhibits protein synthesis, leading to
cell death

Antibiotic mechanism of action