LECTURE 18: TRANSLATION

Replication and transcription processes

o Replication: replicating nucleic acid
o Transcription: initial stage of protein synthesis, copying of segment of DNA into mRNA
Translation, follows transcription
Taking mRNA, information communicated via mRNA, with help of ribosome is converted into protein
Translation: RNA guided polymerization of amino acids into polypeptide sequences
o Guided: not random
o Take individual aas sand converts them into peptide sequences
Amino acids dont spontaneously polymerize, it is thermodynamically unfavorable
o Peptide bond has a higher free energy
o Takes each amino acid and activates it
o Uses ATP and amino acids
Activated amino acid is not immediately used
Transferred to tRNA, shuttle that delivers AA to ribosome
Activation of AA and loading on to tRNA
o TWO STEP PROCESS
o Facilitated by aminoacyl trNA syntehtases
o Synthetases used by ribosome, ribozyme, and results in polymerization of AA
Genetic code
o What combination of nueclic acids encodes for a particular amino acid
Inside the cell:
o Transcription: occurs in nucleus
o Translation: occurs in RER
Right outside the nucleus, layers of membranes sprinkled with ribosomes
o Transcription occurs in nucleus, mrna is sent from nucleus to RER
Ribosome concists of large and small ribosomal subunits, two major building blocks
o Interphase between large and small rests tRNA molecules on mRNA strand
How many proteins are involved in translation mechanism?
o In humans, entire machinery consists of 300 different components
o Protein synthesis is a major investment by cell
Making 300 different components is a significant investment of energy
Around 1/3 of all energy produced by cell goes towards protein synthesis
Structural roles, receptors, catalysts
Major investmenet, crucial to cell survival
Two challenges for protein synthesis:
o 1. The cell must have a mechanism in place to take individual AAs and reproducibly polymerize
them into a specific polypeptide sequence
What are the consequences of innacurate polymerization?
Results in things like amyloid fibril rather than correct prion proteins
The fact were alive is evidence that the cell actually does this really well
o 2. The chemistry, indivudall AAS do not want to polymerize
Must make it happen, and make it happen fast
Challenge 1:
o The genetic code
o All starts with mrna
3 key elements on mrna
o 1. RBS/ shine-dalgarno sequence: ribosomal binding site
Docking site for ribosomal to assemble before it can initiate translation
What is the starting point for replication? ORIs

What is the starting point for transcription? PROMOTOR

o Region in dna that encodes for RNA polymerase binding site
o Remember, it is not transcribed, so it is not mRNA
o It would be hypothetically upstream from RBS
Also characterized by purine richness (As and Gs)
o 2. AUG Start codon
Codes for methionine, whats special about Start Codon?
What distinguishes it from other augs?
Its proximity to the RBS
5-10 nucleotides downstream from RBS, ribosome knows the methionine is
special
Proximity effect
Most organisms use methionine, evidence of common ancestor
o 3. Stop Codon
Small subset of codons, makrs the end of peptide sequence message
Terminates translation, make ribosome pop off
Start and stop codon sets the boundaries of which nucleic acid sequence is actually being read by the
ribosome
o AUG goes in triplets AUU UAG (stop codon)
o Stops at the nucleoside triplet that preceds the stop codon
o Stop codon does not encode for amino acid
TAA, TAG, TGA will cause termination
o In principle, there are 64 different combinations of three nucleotides
But there are only 20 amino acids, more combinations than information to encode for
But, multiple codons can encode for the same amino acid: the degeneracy of the genetic
code
Not every nucleotide triplet encodes for a unique amino acid, many triplets will
encode for the same amino acid
Two types of degeneracy/redundancy of genetic code
o 1. The fact that is a result from third position of nucleotide
o 2. Serine can be AT or AG, 6 different codons, 4 are a result of third nucleotide position
Also arginine, has 6 codons
Where do degeneracies come from?
o Degeneracy originating from completely different codons coding for the same amino acid
Utilization of genetic code in different species
Percentage of codons used in humans vs. e. coli
Humans: we use all six arginine codons with same frequency
E. Coli: only uses primarily 2 of six possible arginine codons
Species preference increases degeneracy, bias results from particular tRNA that is not
produced
o Wobble base pairing, also increases degeneracy, outer most ring (variability factor)
tRNA binds to mRNA, h-bonds between codon and anticodon
In a steric perspective: the structural/orientation positioning is imperfect
2/3 form perfect base pair
Third cannot form perfect h-bond interaction, leads to wobbling/ambiguity
o H-bonding in 3rd base pair are not consistent with perfect WatsonCricke base pairing (A-T/U, G-C)
o This third position is less relevant
Only first two positions form strict interactions, third is a wobble
Between wobble base-pairing and organism specific utilization of genetic code, cell
doesnt just have degeneracy, it CHOOSES to use subset of genetic code

2nd Challenge is actual chemistry

o Amino acid with low free enetery
o Peptide bond with high free energy
o Goes through activated amino acid intermediate
o First half is broken down in to two steps:
1. Individual aino acid is activated with ATP
2. Is then transferred onto a tRNA
These two chemical reactions are facilitated by tRNA synthetase
tRNA synthetase have ability to bind amino aj.cid, ATP, and tRNA
o SPECIFIC FOR AMINO ACIDS
o For every naturally occurring amino acid, there is ONE specific trNA syntehtase
20 amino aicds = 20 tRNA synthetases
What goes on?
o 1st Step: Binding of ATP and specific amino acid for tRNA synthetase
Bind in enzyme active site
Amino acid is activated via ATP, forms adenosyl amino acid intermediate, releases
pyrophosphate which is hydrolyzed (makes reaction irreversible)
o 2nd Step: once activated amino acid sites in active site, tRNA comes and binds
Theres a reason why tRNA synthetases have a specific shape
Binds with two parts of tRNA binds with bottom end of L-stick and top end of L-stick
Top end: binds near activated amino acid, where amino acid is docked on
Bottom end: where tRNA synthetase recognizes the anticodon, and ensures
correct tRNA molecule is bound to the enzyme
After binding of tRNA the chemistry is very simple
o The activated amino acid is transferred onto tRNA, specifically to the 3 end of trNA, the
CCA/CAA end
The 3 end of an adeonisine base, puts amino acid onto 3 hydroxyl, esterifies
Esterifies 3 hydroxyl group on adenoxine by coupling it with the carboxylic acid group
of amino acid
o Once this happens, the amino acid activated form is docked to the trNA, and is ready to travel to
the ribosome
In the ribosome, is where the actual polymerization takes place
o Before synthesis is possible, all of the components must first be brought together
o Ribosome must be assembled onto the mRNA
Starts with ribosomal binding sit, shine-dalgarno sequence
How does the ribosome recognize the RBS?
The ribosome is also RNA, there is a particular region within SMALL
SUBUNIT that is also single-stranded
The Single stranded regions are COMPLEMENTARY
Temporarily forms a double-stranded RNA sequence with mRNA RBS
Translation is initiated by small subunit, 30S (swimberg unites)
The small subunit docks to the mRNA with help of RBS
Just happens that by binding, by forming temporary double stranded RNA, the
two active sites are positioned precisely over the start codon
o Thats why its so important that the start codon, AUG, is 5-10
nucleotides within the RBS
Turns out that this process does not happen by itself
o In order to amek process more efficient there is a number of proteins
that are involved
IF1, IF2, IF3, (IF = initation factor)
o It initiates the formation of the ribosomal complex

As soon as small subunit is bound to mrNA, the tRNA with methionine (start codon)
begins associating with mRNA
Protein cofactors, IF2, assists in process
Energy is also spent in the form of GTP hydrolysis
Once methionine sits on initiation complex, the 50S comes in and binds
Now fully assembles initiation complex, protein synthesis can begin, END OF
INITIATION STAGE
Elongation
Right next to start codon, second active site (A site)
Amino acid binding site, as ribosome begins to polymerize, with help of more
proteins and GTP hydrolysis, it brings in appropriate tRNA
o Appropriate determined by complementary to mRNA
Positioning tRNA next to each other, also positions amino acid in close
proximityWhere
o Amno acids are held in close proximity
o Amino acid that binds in A site has a free amino group
o AA in P site has a carbonyl group
o Amino group, good nucleophile, attacks ester linkage between amino
acid and tRNA,
o Peptide is extended by one building block, ends up at A site
tRNA in P site is now spent, one in A site has an additional amino acid
o Translocation, entire ribosome shifts by 3 nucleotide position
o Moving tRNA in A site into P Site, opening A site for new tRNA
Continues until ribosome encounters stop codon
o No tRNA for stop codon
o Instead there is a abort factor, RF, termination factors
o Associate with stop codon, causes polypeptide sequence attached to
tRNA to be cleaved off, terminating peptide synthesis
o Entire ribosomal complex fall apart, large and small subunit dissociate
Structure of ribosome and peptide synthesis
Cross cut of large ribosomal subunit
PT: petidule transfer, where peptide bonds are synthesized
Where chemical bond formation takes place
Tunnel that leads to back door of ribosomal subunit
Newly synthesized polypeptide sequence is funneled through tunnel
Out to TF, the trigger factor
Where newly synthesized polypeptide sequence is handed off into cavity created
by the trigger factor and where protein folding process can take place