2015 化學生物學 Handout Ch. 4-2

Section 4-6 National Tsing Hua University
CHEM5980
RNA translation
• The translation from mRNA to protein is catalyzed by ribosome, a massive molecular
machine. Ribosome efficiently connect amino acid units through aminolysis reaction
following the sequence provide by mRNA.
• The ribosome catalyzed aminolysis reaction is very similar to modern peptide synthesis
reactions, but it is much more efficient in extending the peptide chain and no
protecting group manipulation is required in enzymatic reaction.
CHEM5980
tRNA structure
• Aminoacyl transfer RNA
(tRNA) is responsible for
carrying amino acyl group,
in the form of ester at 2’ or
3’-OH of 3’-end ribose on
tRNA, and recognizing the
codon on mRNA.
• The cargo amino acyl group
can equilibrate between 2’ or
3’-OH, but it is only reactive
on 3’-OH.
CHEM5980
Ribosome composition
• Every typical human cell has around 4 million ribosomes and account for about 80%
of RNA and 5~10% protein in the cell. Mitochondria have their own ribosomes,
which differ to human ribosome in composition.
• Human ribosome is composed of the large 60S and the small 40S subunits. Each
subunits are further composed of rRNA and ribosomal proteins.
(33)
80S ribosome structure

from C. elegans
(47)
Nat Rev Microbiol 2005, 3, 870 Nat Struc Mol Biol 2012, 19, 560
CHEM5980
Ribosome biogenesis
• Ribosome biogenesis involves
all 3 RNA polymerase (Pol) and
over 150 non-ribosomal factors.
• 18S, 5.8S and 28S component
are transcribed as single 47S by
Pol I, while 5S rRNA is
transcribed by Pol III.
• These pre-RNAs are extensively
modified with
pseudouridylation and
methylation, which are essential
for ribosomal function.
• All ribosomal protein are
transcribed by Pol II and
translated in cytoplasm. 18S
rRNA formed the 40S subunit
with 32 ribosomal proteins,
while 28S, 5.8S and 5S rRNA
form large S60 subunit with 47
ribosomal proteins
Nat Rev Mol Cell Biol 2012, 13, 355
CHEM5980
Ribosome biogenesis
• Use N-15 labelled protein for ribosome assembly and add N-14 protein at different time
point to study the kinetic of the assembly. (Pulse-chase)
• Mass spectrometry exploited to measure the ratio between N-15 and N-14.
Nature 2005, 438, 628

CHEM5980
Ribosome biogenesis
• Time-resolved electron microscope exploited to investigate bacterial ribosome biogenesis.
• More than 1 million snapshots taken.
Science 2010, 330, 673

CHEM5980
Ribosome biogenesis
• Combination of thermodynamic dependence map, ensemble kinetic data and single
particle profiling with structural analysis allow the construction of assembly mechanism.
Science 2010, 330, 673

CHEM5980
Ribosome structure
• Bacterial 70S ribosome can be divided into 2 major
subunits: 50S and 30S. The subunits Joints together
through interface RNAs.
• The 30S subunit contains decoding site that mRNA
sequence can be read in 3-sequence block called
codon.
• The 50S subunit is responsible for the formation of
the amide bond connection.
• Both subunits has 3 tRNA binding site, which are
called A-site, P-site and E-site. A-site is for
accommodating the incoming aminoacyl-tRNA. P-
site hold the tRNA that carries nacent polypeptide
chain. E-site are for the deacylated tRNA binding
before released back to cytoplasm.
•
70S ribosome

30S subunit 50S subunit Nature 2009, 461, 1234
CHEM5980
Translation mechanism
• The elongation of peptide is a cyclic process with one amino acid extension per round
• During this process the peptidyl-tRNA at P-site transfers the growing peptide chain to
the aminoacyl-tRNA at A-site. Then the ribosome shifts relative to the mRNA and tRNAs
empty A-site to accommodate the next aminoacyl-tRNA

CHEM5980
Translation mechanism in detail
Nature 2009, 461, 1234

CHEM5980

• Initiation: This process requires three initiation
factor (IF1~3) and formylmethionine loaded
tRNA (fMet-tRNAfMet), which positioned at the P-
site. The initiation may begin with binding of IF3
to 30S, which was separated from 50S after
translational termination, to release the leftover
mRNA and deacylated tRNA from the previous
round. The IF3-30S complex with mRNA IF1 and
IF2 to become 30S initiation complex then join
50S to form 70S initiation complex. And with
unclear mechanism IFs dissociated with the
hydrolysis of GTP. Nature 2009, 461, 1234
• Elongation: The cycle begins with empty A-site and peptidyl-tRNA at P-site. The next
amino acid was delivered in the complex of elongation factor Tu (EF-Tu), GTP and
aminoacyl-tRNA. The decoding ensure the correct EF Tu-tRNA complex at A-site and
hydrolyze GTP to trigger release of the factors and move aminoacyl end of A-site tRNA
to peptidyl transferase center (PTC). Precise position of α-amino group of aminoacyl-
tRNA toward 2’-OH of peptidyl-tRNA accelerates the elongation.
CHEM5980
Nature 2009, 461, 1234

CHEM5980

• Elongation (cont’d): The peptide bond formation has moved nascent peptide chain to A-
site tRNA. To continue the cycle, tRNA and mRNA must move relative to ribosome. The
translocation of 50S occurs spontaneously after peptide bond formation, but the
movement in 30S require the help from EF-G, which changes the 30S conformation upon
GTP hydrolysis. In this process the ribosomal subunits rotate relatively to each other and
allows tRNAs move to a new site.
• Termination: When stop codon on mRNA moves into A-site of ribosome, release
factors (RF) that recognize the stop codons are recruited and cleave nascent
polypeptide. RF1 and RF2 are involved in release the peptide while RF3 binding
induce the conformational change to allow dissociation of the other RFs. The
ribosome then undergoes ribosome recycle factor processing to recycle ribosome
in separate subunits, yielding 50S subunit and the complex of 30S subunit, mRNA
and deacylated tRNA, which require IF3 to dissociate.
Nature 2009, 461, 1234
CHEM5980
Genetic code for translation

• With 4 possible RNA bases available in 3-digit codon, there are 64 combinations to
code 20 universal amino acids. Therefore are redundant codes for each amino acid.
• Although the triplet codon codes are universal
among all life derived from earth, there are
preference for the code used for each amino
acid. For example most common codon
encodes Alanine is GCG in E.coli K12 but AGA
in human.
• UAG, UAA, UGA are stop codons, but
UAG is least used in E. coli.
• mRNA translates to protein in 5’ to 3’
direction, whereas protein are
synthesized in N to C direction.
sense
antisense
CHEM5980
tRNA processing
• After tRNA transcribed, it is cleaved at 5’ end. Then it is spliced
to give the clover-like structure.
• Followed by the addition of conserved 3’ terminal 5’-CCA-3’ by
nucleotidyltransferase for the attachment of aminoacyl group.
• At the end other enzymes modify the nucleic bases.
http://www.nobelprize.org/educatio
nal/medicine/dna/a/translation/trn
a_processing.html
CHEM5980
tRNA processing
• 85 different modification has been identified on tRNA. Examples of some nucleic bases
modifications are listed below.
• Some modification is relatively similar to normal base; however some are more drastic.
• Some of these modifications are involved in the immune response, the modified tRNA
would cause incorrect amino acid specificity or even frame shift in RNA recognition as a
result of retroviral invasion.
CHEM5980
tRNA processing for decoding

• Examples for tRNA nucleic bases modifications are listed below. Position 34 and 37 are
most heavily modified.
• In many cases from the genetic code wheel, the third codon as purine or pyrimidine
gives different amino acid. The anticodon position that matches this 3rd codon is the
position 34. Modification at position 34 are meant to distinguish between purine and
pyrimidine better to make decoding
process stringent.
• Except for Cys and Ser, pyrimidine
ending codon usually read by
modified G at position 34. G34 often
modified to Gm or Q wobble pairing
with U3 and C3.
• Purine ending codon are read by one
tRNA with U34 or two tRNAs with
U34 and C34. U34 usually modified
to xnm5U.
• Position 37 modification stabilizes
first pairing for codon-anticodon at
position 36.
Ann Rev Genet 2012, 46, 69

CHEM5980
tRNA processing
61 codon available, however less than 45 tRNA available for most organisms.
Gly
Ann Rev Genet 2012, 46, 69

CHEM5980
tRNA processing functions

Decoding genetic codes
Structure maintaining:
• tRNA folding into L-shaped tertiary structure. The major interactions occurs at the
elbow region, where loop T and loop D meet.
• Nucleic base modification can participate in structure stabilization. Methylation is one
popular modification that destabilize Watson-Crick interaction and lead to global
structural change. Other factors such as Mg2+ and polyamine can be altered by tRNA
modification.
Recognition of tRNA:
• Every tRNA synthetase recognize cognate tRNA. Modification
at A loop affect the efficiency of tRNA synthetase.
• The tRNA-EF recognition also altered by tRNA modification.
Translation alteration:
• Lack of few modifications associates with increased frameshift.
Cell growth
Required for translating specific proteins Variable loop
A loop
CHEM5980
tRNA synthetase
• tRNA brings its cargo amino acid to ribosome according to its matching anticodon. The
specificity actually derives from the enzyme that put amino acid and tRNA together:
tRNA synthetases.
• tRNA synthetases recognize the tRNA and the amino acid before joining them, if
mistaken, it would just hydrolyze the product aminoacyl tRNA as proofreading
mechanism.
• High fidelity are required for tRNA synthetases to avoid mistakes in translation. tRNA
synthetase relies on induced fit strategy that adjust both conformation of enzyme and
tRNA for recognition. tRNA synthetase recognizes all tRNAs that code for the same
amino acid.
CHEM5980
tRNA synthetase
Mol BioSyst 2013, 9, 343-351

CHEM5980
Non-translational function of tRNA synthetase

• Higher eukaryote tRNA synthetase
comprise of a catalytic module, a RNA
binding module and a new domain.
• tRNA synthetases have functions other
than translation. These functions can be
broadly divided, but not limited, as
following:
• Mediation of amino acid and glucose
metabolism
• Initiate or silence inflammatory
response
• Cell death and stress response
control
• Amplify or inhibit immune response
• Regulation for development of new
tissue or organ
• Controlling balance of angiogenesis
Nat Chem Biol 2013, 9, 145

CHEM5980
Start point and end point of translation

• mRNA molecules has structure features to inform ribosome the region of translation.
Ribosome binding site (RBS) hybridize with complementary sequence on ribosome to
begin translation.
• The translated region always
begin with start codon AUG
(methionine) and ends before
stop codon.
• The first few amino acids in the
translated protein are modified
by enzymes. In prokaryotes,
formyl methionine, the formyl
group on the first amino acid
can be removed by peptide
deformylase, whereas
eukaryotic methionine can be
removed by aminopeptidase.
CHEM5980
Internal ribosomal entry sequence (IRES)

• RBS helps ribosome attachment to mRNA and begin translation, and this process is
tightly controlled under different mechanisms.
• Initiation factors (IF) are required for translation process to begin in eukaryotes.
G-cap at the 5’ end of mRNA directs ribosome to RBS by interaction with IF4E to
begin translation.

CHEM5980

• Another mechanism for ribosome binding and translation uses internal ribosomal entry
sequence (IRES), which is rare for mammalian cells but widely used by virus.
• The IRES by-passes the host translation regulations. Therefore, when virus hijacks the
host protein production, host cells are not able to shut it down.
• Initiation on type 1 and type 2 IRESs involves their specific binding to the eIF4G, which is
enhanced by eIF4A.
• The eIF4G–eIF4A complex recruits 43S complexes to type 1 and type 2 IRESs without the
involvement of eIF4E.

CHEM5980

• Type 3 IRESs involves their interaction with the eIF3 and 40S subunit components
of 43S complexes and on type 4 IRESs involves their binding to 40S subunits.
• Type 3 IRESs directly attach 43S complexes to the initiation codon independently of
eIF4F, eIF4B, eIF1 and eIF1A, whereas type 4 IRESs initiate without eIFs or tRNAMet
(the P-site of the 40S subunit is occupied by an IRES domain that mimics codon–
anticodon base pairing). Therefore, IRES-mediated initiation might be resistant to
cellular regulatory mechanisms.
Target P-site

CHEM5980

• While virus use IRES for
invasion it has become a
good target to treat viral
infection.
• Human ribosome do not
mutate with virus,
therefore the IRES must
be somewhat conserved.
• Inhibitors for HCV IRES Nat Chem Biol 2009, 5, 823
have been developed,
structural study have
shown how it worked for
inhibition.
PNAS 2012, 109, 5223

CHEM5980
Translation control
• Cell controls protein translation by the slow, rate-limiting, initiation step. Such control
can be performed with different mechanisms.
• In the case of E. coli heat response proteins, they are constitutively transcribed. But
only when the temperature rises that allow mRNA unfold to expose the RBS can the
translation begin.
• Regulatory proteins can
also control translation
by binding to the mRNA
segment that inactivate
the RBS.
• Alternatively, regulatory
RNA can hybridize
mRNA to mask the RBS.
Or a protein can bind to
mask RBS. Both strategy
prevent the binding of
ribosome and inhibit
protein translation.
CHEM5980
Translation fidelity
• Aminoacyl tRNAs are substrate to ribosome but they do not engage the translation on
their own, instead, other proteins called elongation factor (EF) participated.
• In human cells, EF-1α forms complex with GTP and tRNA and bind reversibly to ribosome.
If anticodon match the codon on mRNA, EF-1α catalyze GTP hydrolysis and release
aminoacyl tRNA to allow delivery of amino acid. EF-1β help remove GDP from EF-1α so it
can continue the catalytic cycle.
• Another protein EF-G resemble the
appearance of tRNA- EF-1α complex and
removed tRNA from the active site. The
hydrolysis of GTP catalyzed by EF-G
pushes nascent peptide and
mRNA forward.
EF-1a +
EF-G aminoacyl tRNA
CHEM5980
Ricin toxicity
• Ricin is a natural product from castor beans.
• Ricin is one of the most toxic molecules on the planet with LD50 around 0.022mg/kg.
• Ricin consists of α chain and β chain, where the β chain is responsible for delivery into
mammalian cell through carbohydrate binding and the α chain is responsible for
targeting ribosome.
• The Ricin α chain has rRNA N-glycosylase activity that
inactivate ribosome by depurinate adenine at A4324,
which is essential for EF-1 and EF-2 binding to ribosome.
• One Ricin molecule can catalyze depurination of 1500
ribosomes in one minute, which makes it so toxic.
α chain β chain
Cell 2010, 141, 222
CHEM5980
Ricin antidote
• Compounds screened to find small molecules that protect ricin toxicity.
• Ilimaquinone (IQ) is a marine product causes breakdown of the Golgi apparatus and its
fragmentation into small vesicular structures.
• Brefeldin A (BFA) is a fungal metabolite that disrupts the structure and function of the
Golgi apparatus, and strongly impairs intracellular protein transport and secretion.
• Studies show that Retro-1 and Retro-2 inhibit retrograde toxin transport between early
endosomes and the trans Golgi network (TGN)/Golgi complex.
Toxins 2012, 4, 15
CHEM5980
Riboswitch
• The recognition of small molecules in physiological systems is usually performed by
proteins because 20 amino acids allow formation of complicated receptors.
• Until lately, it was discovered the some mRNA has structured domains in the
untranslated regions to selectively bind small molecules and alter the outcome of
translation. This mechanism is called riboswitch.
• Typical small molecule binding causes conformational change in the 5’ untranslated
region and shut translation of downstream RNA sequence by either formation of
terminator or masking the RBS.
CHEM5980
Structural analysis of riboswitch by in-line probing

• To understand how small molecules bind
to riboswitch, a method called in-line
probing can be exploited. This method
use 2’-OH on ribose to cleave RNA
backbone and it is active when 2’-O and
5’-O from next ribose form in-linear
conformation. As the result, the highly
structured region of RNA is not cleaved.
• Coenzyme B12 riboswitch is a well
studied example. Rapid cleaved locations
in yellow. Upon coenzyme B12 binding,
decreased and increased location are in
red and green respectively.

CHEM5980
Riboswitch
• Small metabolites are
usually ligands to control
the riboswitches.
Different ligands require
their own riboswitch.
• Some common ligands coenzyme B12 flavin
thiamine
mononucleotide
included: coenzyme B12, pyrophosphate
thiamine pyrophosphate
(TPP), guanine/adenine,
flavin mononucleotide
(FMO), glucosamine 6-
guanine / adenine
phosphate (GlcN6P), S- S-adenosylmethionine
adenosylmethionine
(SAM) and lysine.
lysine GlcN6P
CHEM5980
Engineering riboswitch
• Riboswitch controls translation via small molecule, this strategy can be useful in
control recombinant protein expression.
• Need to develop unnatural ligand to avoid interference from natural ligands.
• To change selectivity from adenine to other compounds, U47 and U51 of the RNA are
modified to develop orthogonal selective riboswitch.
Watson-Crick-like
interactions are easier
to predict/modify
PNAS 2010, 107, 2830

CHEM5980
Site-specific incorporation of functional groups to protein

• Proteins have their specific functions and specificities toward their substrates.
These properties arise from their unique folded structures.
• Site specific modification on protein can help our understanding for a specific
protein. As example, site-specific introduction of certain unnatural functional
groups can help reveal structure and elucidate function.
• With protein structure available, artificial functional groups can be incorporate
at specific site to alter the protein function in a tailor-made fashion.
• However such site-specific modification using functional groups beyond those
from 20 natural amino acid are especially challenging. Some unnatural amino
acid and their utilities are listed below.
CHEM5980
Translation of unnatural amino acid

• Cellular machinery have been exploited to produce protein for its efficiency. Is it
possible to produce artificial protein with custom amino acid?
• The 64 combinations of codons have been used to encode 20 amino acids and the stop
signal. Where to find a special codon for the custom amino acid?
• Many species incorporate selenocysteine (Sec) as the 21st amino acid. Interestingly, the
stop codon UGA was used.
CHEM5980
In vitro translation of unnatural amino acid

• Early studies to incorporate unnatural Evolved
amino acid use amber suppressor ribozyme
tRNA, which recognize the lease
common stop codon UAG. Chemical
conjugation of unnatural amino acid
to the amber suppressor tRNA allows
recognition
the in vitro synthesis on protein with
unnatural amino acids.
• As an improvement ribozyme can be

loaded to resin and allow user to
charge unnatural amino acid to tRNA
with ease. The product aminoacyl-
tRNA can then be used in the in vitro
translation to produce unnatural
protein.
Methods 2006, 36, 239
CHEM5980
From in vitro to cell-based translation

• Cell-based translation of unnatural amino acid (UAA) require the tRNA to be
biosynthesized in the cell. The advantages of this strategy over the in vitro method
include: technically easier after the clone has been established and higher yield
because the aminoacyl tRNA is chemically synthesized and do not re-acylate in cell with
the in vitro method.
• Cell-based UAA translation requires evolve of new tRNA synthetase that fulfills:
1. The new tRNA translate only the codon encode the UAA
2. The UAA cannot be substrate of any endogenous tRNA synthetase
3. The evolved tRNA synthetase only recognize the orthogonal tRNA but not endogenous
tRNAs
4. The new synthetase only catalyze aminoacylation of cognate tRNA with UAA
natural
1 4
Must not Must not
unnatural
2 3
Must Must Must
tRNA amino acid
codon tRNA
synthetase
CHEM5980
From in vitro to cell-based translation

• To construct a cognate system
following must be considered:
1. Codon: use Amber stop codon UAG.
2. tRNA: to avoid recognition by
endogenous tRNA synthetase, used
tRNA from other organism. Red
bases are those involve in
recognition. Yeast and M. jannaschii
(archaea) are candidates.
3. tRNA synthetase: Just use the pair
from other organism to avoid
unnecessary evolution.
4. Unnatural amino acid: structurally
not far from existing amino acid are
more likely to succeed. Need to
evolve the tRNA synthetase to take
the UAA and transfer to cognate
tRNA.
Methods 2005, 36, 227

Science 2001, 292, 498
CHEM5980
Evolve and screening

• M. jannaschii Tyr tRNA synthetase has least contact to tRNA anticodon loop therefore
has better chance to change coding to CUA. So M. jannaschii Tyr tRNA, synthetase pair
were selected.
• Some activity found between M. jannaschii tRNACUATyr and E. coli tRNA synthetase,
therefore mutations at 11 sites on tRNA were performed.
• Negative selection removes tRNA recognized E. coli tRNA synthetase, positive selection
remove those tRNA not recognized by M. jannaschii Tyr tRNA synthetase.
Designed
mutation
sites
Evolved
mutation
sites
2
Methods 2005, 36, 227
CHEM5980
Evolve and screening

• M. jannaschii Tyr tRNA were mutated to orthogonal to E. coli tRNA synthetase.
• Next step is to mutate M. jannaschii Tyr tRNA synthetase to transfer supplied UUA to
tRNA. In positive selection strains that tRNA synthetase incorporate UAA or endogenous
AA survive. In negative selecting no UAA provideed, only those transfer UAA survive.
Ann Rev Biochem 2010, 79, 413

CHEM5980
tRNA suppressing tech (Pete)
Ann Rev Biochem 2010, 79, 413

CHEM5980
Bacterial ribosome and antibiotics

• Ribosome is a good target for
antibiotic development because
protein synthesis is essential for
all organism and many part of
the ribosome is conserved
through the evolution.
• A large number of natural
antibiotics kill bacteria by
disrupting the functions of
ribosome.
30S 50S
Nat Rev Microbiol 2005, 3, 870
CHEM5980
Examples of antibiotics
• Protein factors are also targets for antibiotics such as kirromycin and fusidic acid.
• Erythromycin belongs to macrolide family while streptomycin belongs to
aminoglycosides.
CHEM5980

DM: drug modification
TM: target mutation
E: efflux/membrane
permeability
TA: target alteration
FP: factor-assisted
protection

CHEM5980
Tetracycline
• Tetracycline functions by preventing aminoacyl-tRNA from binding to A-site on 30S
subunit of ribosome, therefore stop peptide from being synthesized by ribosome.
• Bacteria has evolved resistance with different strategies. tetM gene encodes for
ribosome protecting protein, which binds to ribosome and prevent tetracycline
binding to ribosome. In addition, bacteria expresses efflux pump to remove
tetracycline from cytoplasm.
• Tetracycline binds to both bacterial and mammalian ribosomes. However as
tetracycline can be import into bacteria by pump, which is not found in mammals,
mammals are less affected.
50S
30S
PNAS 2012, 102, 16900 Cell 2000, 103, 1143

CHEM5980
Macrolide antibiotic
• Newly synthesized peptide passes through 50S subunit tunnel. The narrowest point is
where ribosomal protein L4 and L22 merge with rRNA to control the synthesis of
certain peptides.
• Macrolides are natural polyketide compound that consist of 14~16 membered-lactone
ring with substituents. Macrolides functions as antibiotics by binding to the peptide
tunnel on 50S to inhibit the elongation of nascent peptide.
• Resistance toward macrolide has emerged: the mutations at nucleotide near 2058 alter
the binding site for macrolides. Mutation around nucleotide 752, protein L4 and L22
changes the hairpin structure that limit the narrow passage in the tunnel.
nascent
peptide
chain
50S
Biochemistry (Moscow) 2010, 75, 1501 Nat Rev Microbiol 2014, 12, 35
CHEM5980
How antibiotics kill bacteria

• There are 5 major antibiotic targets or pathways.
B-lactam
Vancomycin
Macrolide
Aminoglycoside
Tetracycline
Daptomycin
Surfactin
Quinolone
Aminocoumarin
Sulfa drugs Rifamycins
Folate analog Fidaxomicin
J Antibiotics 2014, 67, 7

CHEM5980
Timeline of antibiotics
• In the past 80 years, the discovery of antibiotics mainly from two routes. One
is identification of natural antimicrobial compounds and the other is through
man-made molecules.
• The major identification of natural antibiotics occurred in 40’s to 60’s. Current
new generations of modified antibiotics actually based on the same scaffolds
from the natural compounds.
• Antibiotics with new scaffolds are in urgent need to avoid bacterial resistance.
CHEM5980
Antibiotic resistance
• Antibiotic can viewed as a special

group of drug that when misused
not only the patient but the broad
community will be affected.
• The antibiotic-resistant bacteria rise
due to the therapy (selection).
Therefore the drug-resistance
Last resort of emergence is not matter of “if” but
treatment
“when”.

CHEM5980
Antibiotic scaffolds
• Antibiotics development through modification of existing scaffold.
• Generations of antibiotics share same core structure

CHEM5980
Some antibiotic resistant bacteria

MRSA: VRE
Methicillin-resistant Staphylococcus aureus or Vancomycin-Resistant Enterococcus
Multiple-resistant Staphylococcus aureus
Spellberg, Brad. Rising Plague: the Global Threat from

Deadly Bacteria and Our Dwindling Arsenal to Fight Them.
Amherst, NY: Prometheus, 2009. Print.
CHEM5980

CHEM5980
Cost for new antibiotics development

• Typical drug development timeline for new antibiotics are shown below. Drugs
candidates take more than a decade to be screened and pass though a
number of stages to reach new drug. Cost for this process is also very high.

CHEM5980
Some new antibiotic scaffolds

• Improved screen methods have provided some new scaffolds for development
of new antibiotics. Some were found from synthetic libraries such as TK-666,
others were identified from natural sources.
bacterial
thymidylate kinase
Block fatty acid

biosynthesis
DNA gyrase and

topoisomerase IV
inhibitor of the bacterial type II

p-aminobenzoate J Antibiotics 2014, 67, 7
topoisomerase
(folate involved) inhibitor
biosynthesis
CHEM5980
DNA and RNA library

• With the advances in DNA synthesis, making DNA library is
straightforward: just use the mixture of all four nucleotides
building block at desired position. The random DNA library
usually flanked with non-variant sequence to allow PCR
amplification.
• DNA library can be easily expanded to RNA and protein
library with transcription and translation by cellular
machinery. Alternatively, single strand DNA can also form
specific structures controlled by Watson-Crick pairing and
other interactions. These non-double helix structures able
to interact with variety of molecules are called aptamers.
• In contrast to single strand
DNA, which has no significant
biological functions, RNA
library can mimic their natural
counterpart such as
riboswitch. This type of library
can be transcribed from DNA
library by reliable T7 RNA
polymerase.
CHEM5980
From RNA library to drug Pu

• Pegaptanib, the first approved
aptamer drug (2004), is
developed to treat age-related
macular degeneration (AMD).
• Pegaptanib is a single strand RNA
molecule that targets vascular
endothelial growth factor (VEGF)
to inhibit the growth of blood
vessel. Py
• Pegaptanib was developed with
SELEX technology in creating a
RNA aptamer library.
• To increase the in vivo stability,
2’-OH of ribose on RNA was
methylated or replaced by
fluoride. 2’ OH
Pu 2’ OMe
Py 2’ F
Nat Rev Drug Disco 2006, 5, 123

CHEM5980
RNA library for ribozyme

• RNA library can be used directly in screening for its enzyme activity. In one example, 76
random RNA sequences library were made to screen for glycosylation activity.
• This screening uses 4-thiouracil, an analog of uracil, to test the glycosylation at 3’ end.
If glycosylated, the thiol functinality would allow attachment of RNA molecule to
mercury, which is loaded on gel.
• Isolation of the selected
RNAs would allow signal
amplification through
reverse transcription to
DNA and followed by
PCR.
• Selected hit provide as
much as 10 million fold
of increase in
glycosylation activity
than background.
CHEM5980
Ribozyme and RNA world

• RNA world hypothesis suggest that RNA is the molecule in early era.
• Such hypothesis originated from the evidence that RNA stores genetic information like
DNA, and catalyzes chemical reactions (ribozyme) like protein enzymes. Therefore, it
may have played a major role in the evolution of cellular life.
• In addition to the RNA world hypothesis, where does the predominance of single
enantiomer over its enantiomer arise?
• One theory has suggested that a right-handed RNA enzyme emerged with the capacity
to make copies of other right-handed RNA molecules, including itself, but ignores left-
handed L-RNA. But is it really the case?
Nat Chem 2013, 5, 360

CHEM5980
Ribozyme with cross-chiral activity 1~10 rounds
L substrate
• Random D-RNA library made to screen for L-RNA
ligation activity. Totally 16 round of mutation-selection D enzyme
performed to find a good RNA ligase that can function D enzyme 11~16 rounds
on the opposite but not the same chirality.
• This chirality setup (D-enzyme with L-substrate) avoids
Watson-Crick pairing because opposing enantiomers of
RNA do not form contiguous base pairs. Therefore the
L substrate
enzyme functions purely by its tertiary interactions as
other protein enzymes.
• 83 nucleotide D-RNA found as functional ligase.
Nature 2014, ASAP

CHEM5980
Ribozyme with cross-chiral activity

• The ligase function of the selected ribozyme can be expanded to synthesize longer
RNA of the opposite chirality from shorter RNA fragments.
• Interestingly, when the template is the D form
sequence of the L-ribozyme, the ribozyme
catalyzes the replicate itself in the opposite D
form, that is, a enzyme that makes its own
mirror image!
• The evolution may have cross-chiral
polymerization of RNA in certain era that both
chirality were indispensable. However
subsequent key evolution may arise in one
chirality turn the evolution toward that one.
L enzyme 30 nt
D substrate
Nature 2014, ASAP

CHEM5980
From RNA to protein library

• RNA library can be readily
translate into peptide/protein
library, but the problem is the
active peptides/proteins are
cannot be easily sequenced to
tell the identity.
• A clever strategy use puromycin
to help identify the translated
peptide/protein.
• Puromycin is an antibiotic that
functions by inhibit translation
at ribosome. It mimic the 3’-end
nucleotide of aminoacyl-tRNA,
which brings amino acid unit to
ribosome, except the nascent
peptide cannot continue
elongation due to its unreactive
amide rather than ester.
CHEM5980
mRNA display
• When puromycin is covalently attached
to 3’ end of mRNA, it move toward
ribosome as the translation progresses.
Puromycin eventually inhibits peptide
synthesis and forms and peptide-mRNA
hybrid molecular.
• As the protein-encoding mRNA is
attached, it serves as label to protein
and allows amplification for
identification.
• In vitro analogous of phage display but
with several advantages:
1. Library size for the virus-based display
systems is limited by the transformation
efficiency of virus.
2. Avoids unwanted selection pressure,
such as poor protein expression, and
rapid protein degradation.
PNAS 1997, 94, 12297
CHEM5980
mRNA display in practice

• In practice, mRNA display
would include selection of
peptide/protein functions.
• DNA library transcribed to
RNA and treated with DNase.
• Conjugation of puromycin-
containing linker by enzyme
or UV cross-linking.
• High Mg2+ or K+ condition to
promote puromycin reaction.
• Reverse transcription.
• Functional screen and
isolation
• Amplification and sequencing.
Nat Proto 2011, 6, 1163

CHEM5980
mRNA display and macrocyclic library

• In addition to the natural peptide/protein library, the in vitro setting allows the
mRNA display to introduce more diversity.
• Unnatural amino acid can be incorporated through “protein synthesis using
recombinant elements” (PURE) technology. In addition, chemical modification such
as cyclization of the peptide chain are compatible with mRNA display. These
strategies create more unique peptides that are cannot evolve from nature.
Drug Disco Today 2014, 19, 388

CHEM5980
mRNA display and macrocyclic library

• Genomic DNA appears in double helix to store information. However in cyclic
Drug Disco Today 2014, 19, 388

2015 化學生物學 Handout Ch. 4-2

Uploaded by

Document Information

Copyright

Available Formats

Share this document

Share or Embed Document

Sharing Options

Did you find this document useful?

Is this content inappropriate?

Copyright:

Available Formats

2015 化學生物學 Handout Ch. 4-2

Uploaded by

Copyright:

Available Formats

Section 4-6 National Tsing Hua University

80S ribosome structure

Nature 2005, 438, 628

Science 2010, 330, 673

Science 2010, 330, 673

Nat Rev Mol Cell Biol 2008, 9, 242

Nat Rev Mol Cell Biol 2008, 9, 242

Translation mechanism in detail

Nature 2009, 461, 1234

Translation mechanism in detail

Translation mechanism in detail

Nature 2009, 461, 1234

Translation mechanism in detail

Genetic code for translation

tRNA processing for decoding

Ann Rev Genet 2012, 46, 69

Ann Rev Genet 2012, 46, 69

tRNA processing functions

Mol BioSyst 2013, 9, 343-351

Non-translational function of tRNA synthetase

Nat Chem Biol 2013, 9, 145

Start point and end point of translation

Internal ribosomal entry sequence (IRES)

Nat Rev Mol Cell Biol 2010, 10, 113

Internal ribosomal entry sequence (IRES)

Nat Rev Mol Cell Biol 2010, 10, 113

Internal ribosomal entry sequence (IRES)

Nat Rev Mol Cell Biol 2010, 10, 113

Internal ribosomal entry sequence (IRES)

PNAS 2012, 109, 5223

Structural analysis of riboswitch by in-line probing

Nat Rev Mol Cell Biol 2004, 5, 451

PNAS 2010, 107, 2830

Site-specific incorporation of functional groups to protein

Translation of unnatural amino acid

In vitro translation of unnatural amino acid

• As an improvement ribozyme can be

From in vitro to cell-based translation

From in vitro to cell-based translation

Methods 2005, 36, 227

Evolve and screening

Evolve and screening

Ann Rev Biochem 2010, 79, 413

tRNA suppressing tech (Pete)

Ann Rev Biochem 2010, 79, 413

Bacterial ribosome and antibiotics

Bacterial ribosome and antibiotics

Nat Rev Microbiol 2014, 12, 35

PNAS 2012, 102, 16900 Cell 2000, 103, 1143

How antibiotics kill bacteria

J Antibiotics 2014, 67, 7

• Antibiotic can viewed as a special

J Antibiotics 2014, 67, 7

J Antibiotics 2014, 67, 7

Some antibiotic resistant bacteria

Spellberg, Brad. Rising Plague: the Global Threat from

Bacterial ribosome and antibiotics

Nat Rev Microbiol 2014, 12, 35

Cost for new antibiotics development

J Antibiotics 2014, 67, 7

Some new antibiotic scaffolds

Block fatty acid

DNA gyrase and

inhibitor of the bacterial type II