How cells read the genome from DNA to protein Notes

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Part II Chapter 6 How Cells Read the Genome: From DNA to Protein
Central Dogma: DNA to RNA to Protein
RNA Molecules:
• Using ribose sugar instead of deoxyribose
• Uracil base instead of thymine base
• single-stranded
• can fold into 3D shape just like polypeptide
Transcription:
• RNA polymerase
• Substrate: ribonucleoside triphosphates (ATP,
CTP, UTP, and GTP)
• hydrolysis of high-energy bonds provides the
energy needed to drive the reaction forward
• RNA polymerases can start an RNA chain
without a primer (errors in RNA is not that
signiﬁcant compared to DNA)
• RNA polymerases have a modest
proofreading mechanism
• just behind the region where the
ribonucleotides are being added, the RNA
chain is displaced and the DNA helix re-forms
Bacterial Transcription:

• sigma (σ) factor associates with the core enzyme and assists it in reading the signals in the
DNA that tell it where to begin transcribing
• σ factor and core enzyme are known as the RNA polymerase holoenzyme
• holoenzyme slide along the DNA sequence to find a starting signal called promoter
• σ factor then binds tightly to the promoter
• holoenzyme opens up the double helix and the core enzyme starts to transcribe RNA sequence
• However, holoenzyme is still connected to the promoter, so the short RNAs are often released,
forcing the core enzyme to start over.
• Eventually this process of abortive initiation is overcome, and core enzymes discard the σ
factor as well as the connection to promoter
• Polymerase continue to transcribe in a stepwise fashion
• The enzyme encounters a terminal signal called terminator
• Terminator is a DNA sequence which consists of A-T nucleotide pairs, when the terminator is
transcribe into RNA, the RNA will automatically folded into the hairpin shape, thereby stop the
polymerase and release it from the DNA sequence
• Core enzyme is released and reassociate with σ factor to form a holoenzyme
Eukaryotic Transcription:
• RNA polymerases I and III transcribe the genes encoding transfer RNA, ribosomal RNA, and
various small RNAs. RNA polymerase II transcribes most genes, including all those that encode
proteins, and our subsequent discussion therefore focuses on this enzyme.
• Bacterial RNA polymerase only needs a single transcription factor: σ factor; while eukaryotic
RNA polymerase requires many factors, collectively called general transcription factor.
• Eukaryotic transcription must take place on DNA that is packed into nucleosomes and high-
order forms of chromatin structure.
• General transcription factor are needed for all promoters that RNA polymerase II uses. They
are used to imitate the transcription and release the polymerase form the promoter to start
transcribing.They are: TFIIA, TFIIB, TFIIC, TFIID, and so on (TFII standing for transcription
factor for polymerase II).
• TFIID binds to a short DNA sequence composed of the A and T, which is called a TATA
box. The subunit of TFIID called TBP (TATA-binding protein) recognise the TATA box
and binds to it.
• The binding of TATA box causes the distortion in the DNA, it brings the DNA sequence
on the both side together and creates a physical landscape of the promoter.
• Other factors then assemble, along with the RNA polymerase II, to form a complete
transcription initiation complex.
• TFIIH, which contains DNA helicase as one of its subunits, unwind the DNA double
helix, exposing the template strand.
• Like bacterial RNA polymerase, RNA polymerase II also synthesises a short length of
RNA and performs conformational changes before releasing form the promoter and
start transcribing.
• In this process, phosphate group is added to the tails of RNA polymerase II (known as
the CTD or C-terminal domain)
• In human, CTD contains 52 tandem repeats of a seven-amino acid sequence, serine
located at the fifth position in the repeat sequence (Ser5) is phosphorylated by TFIIH
• RNA polymerase II will then disengage from the general transcription factor.
• In addition, transcriptional activators must bind to specific sequences in DNA (called
enhancers) and help to attract RNA polymerase II to the start point of transcription

• eukaryotic transcription initiation in vivo requires the presence of a large protein complex known
as Mediator, which allows the activator proteins to communicate properly with the polymerase II
and with the general transcription factors.
• transcription initiation in a eukaryotic cell typically requires the recruitment of chromatin-
modifying enzymes, including chromatin remodeling complexes and histone-modifying
enzymes, which will increase access to the DNA in chromatin
• RNA polymerase also causes the problem of DNA
supercoiling: In eukaryotes, DNA topoisomerase keeps removing the supercoiling tension; in
bacteria, DNA gyrase uses the energy of ATP to pump the supercoil into DNA
Modiﬁcation of eukaryotic pre-mRNA:
• RNA 5’ end capping: one (a phosphatase) removes a phosphate from the 5ʹ end of the nascent
RNA, another (a guanyl transferase) adds a GMP in a reverse linkage (5ʹ to 5ʹ instead of 5ʹ to
3ʹ), and a third (a methyl transferase) adds a methyl group to the guanosine

• RNA cap helps the cell to distinguish mRNAs from the other types of RNA molecules present in
the cell.
RNA splicing:
• Each splicing event removes one intron, proceeding through two sequential phosphoryl-
transfer reactions known as transesteriﬁcations; these join two exons together while removing
the intron between them as a “lariat”
• Splice site contains a consensus short nucleotide sequence, which acts as a cue for where
splicing is to take place.
• RNA splicing is performed by spliceosome. 5 short RNA molecule: U1,U2,U4,U5 and U6,known
as snRNA (small nuclear RNA), each is then complexed with protein subunits to form an snRNP
(small nuclear ribonucleoprotein). snRNPs then form the core of spliceosome.
• Recognition of splice junction is performed through base pairing between the snRNAs and the
consensus RNA sequences.

• Energy form ATP hydrolysis is used to build up the spliceosomes and break or form the base
pairs between RNA subunits and/or mRNA.
Fidelity of RNA splicing:
• RNA polymerase tail carries several components of spliceosome, and these components are
transferred directly from the polymerase to the RNA as the RNA emerges from the polymerase.
This helps the cell to keep track of introns and exons. It helps the cell to mark the introns.
• Exon definition: Exon size tends to be much more uniform, averaging about 150 nucleotide
pairs. SR proteins assemble on exon sequence and help to mark off splice site and recruit
protein, U1 for downstream and U2 for upstream. SR proteins bind preferentially to specific RNA
sequence in eons, termed splicing enhancers.
• Chromatin structure can alter the speed of transcribing thus splicing. So the speed can be
minimised so exon skipping can be minimised as well.
• Some histone modifications attract components of spliceosome, which will be transferred to
emerging RNA.

3’ end process of RNA:
• 3’ end of each mRNA molecule is specified by signals
encoded in the genome.
• Two multisubunit proteins, called CstF (cleavage stimulation
factor) and CPSF (cleavage and polyadenylation specificity
factor), are on the RNA polymerase tail.They binds to their
recognition sequences on the emerging RNA molecule.
• RNA is cleaved from the polymerase. An enzyme called
poly-A polymerase (PAP) adds and produces around 200
A nucleotides to the 3’ end.
• Poly-A-binding proteins assemble onto the A tail and help
to determine the final length of the tail.
• After the 3’ end of pre-mRNA has been cleaved, RNA
polymerase II continues to transcribes. This unprotected
RNA is then degraded by a exonuclease on the
polymerase tail, which will eventually cause the termination
of transcription.
Export of mature RNA:
• mRNA is distinguished by the protein it has. Cap-binding
protein, exon junction complexes and poly-A-binding protein
marks the completion of capping, splicing and poly-A
addition.
• Improperly processed mRNAs and other RNA debris
(excised intron sequence) is processed and recycled by
nuclear exosome, which is rich of exonuclease.
• Successfully processed mRNAs are guided through the
nuclear pore complexes.(NPCs)
• RNA must pass through the pore by active transport.
Nuclear transport receptor must be loaded onto RNA,
which is performed with 3’ cleavage and polyadenylation.
• Some protein attached on the RNA will travel out of the
nucleus and some protein will remain attached. In addition,
some protein will add onto the mature RNA molecule.

Non-coding RNA:
• ribosomal RNAs (rRNA) is the most abundant RNA in living organism. RNA polymerase I is
dedicated to producing rRNAs. RNA polymerase I is absent of C-terminal, which explains why
rRNA is neither capped or polyadenylated.
• 4 types of rRNAs, 3 (18S, 5.8S, 28S) are made by chemically modifying and cleaving a single
large precursor rRNA., fourth(5S) is synthesised from a separate cluster of genes by RNA
polymerase III.
• There are chemically modification(2’-O-methylated nucleotide, isomers of uridine called
pseudouridine) on precursor rRNA before rRNAs are cleaved out and assemble into ribosomes.
The modification is hypothesised to aid the folding and assembly of the final rRNA.
• Nucleolus is the location where rRNAs are assembled to make
ribosome as well as telomerase, tRNAs and other RNA-protein
complexes.
From RNA to Protein:
• RNA sequence is divided into a group of three consecutive nucleotides,
called codons. One amino acid can be coded with more than one
codon.
• tRNAs are responsible for carrying amino acids. They contain
anticodons which can be paired with the codons on RNA. They are
more than one type of tRNAs for an amino acid. Also,
mismatch(wobble) is allowed for the third base pairing. So a tRNA can
pair with more than one codon.
• tRNA are synthesised by RNA polymerase III. They are covalently
modified before exiting form the nucleus.
• tRNA splicing uses a cut-and-paste mechanism rather than a lariat
intermediate. Modified nucleotide sequence is also found in tRNA,
which affects the conformation and base-paring of tRNA and thereby
facilitate the recognition of the appropriate mRNA codons.
• Recognition and attachment of the correct amino acid depends on enzymes called aminoacyl-
tRNA synthetase. Most cell have different synthethase enzymes for different amino acids.In
bacteria, less than 20 synthetase can be found, which means it can put identical amino acids on
two types of tRNA. One is correct and another needs modification to become correct.

• Most synthetase enzyme choose the correct amino acids in two steps:
1.The correct amino acid has the highest afﬁnity for the active-site pocket of its synthetase and is
therefore favored over the other 19
2.A second discrimination step occurs after the amino acid has been covalently linked to AMP:
when tRNA binds, the synthetase tries to force the adenylated amino acid into a second editing
pocket in the enzyme
• Most tRNA synthetases directly recognize the matching tRNA anticodon; these synthetases
contain three adjacent nucleotide-binding pockets, each of which is complementary in shape and
charge to a nucleotide in the anticodon.
RNA messages are decoded in ribosome

• The small subunit provides the framework on which the tRNAs are
accurately matched to the codons of the mRNA, while the large
subunit catalyzes the formation of the peptide bonds that link the
amino acids together into a polypeptide chain
• a ribosome contains four binding sites for RNA molecules: one is
for the mRNA and three (called the A site, the P site, and the E
site) are for tRNAs
• tRNA binding (step 1), peptide bond formation (step 2), large
subunit translocation (step 3), and small subunit translocation
(step 4). As a result of the two translocation steps, the entire
ribosome moves three nucleotides along the mRNA and is
positioned to start the next cycle.
• In step 1, a tRNA carrying the next amino acid in the chain binds
to the ribosomal A site by forming base pairs with the mRNA
codon positioned there, so that the P site and the A site contain
adjacent bound tRNAs.
• In step 2, the carboxyl end of the polypeptide chain is released
from the tRNA at the P site (by breakage of the high-energy
bond between the tRNA and its amino acid) and joined to the
free amino group of the amino acid linked to the tRNA at the A
site, forming a new peptide bond. This central reaction of
protein synthesis is catalyzed by a peptidyl transferase
contained in the large ribosomal subunit.
• In step 3, the large subunit moves relative to the mRNA held by
the small subunit, thereby shifting the acceptor stems of the two
tRNAs to the E and P sites of the large subunit.
• In step 4, another series of conformational changes moves the
small subunit and its bound mRNA exactly three nucleotides,
ejecting the spent tRNA from the E site and resetting the
ribosome so it is ready to receive the next aminoacyl-tRNA.
• Two elongation factors enter and leave the ribosome during
each cycle, each hydrolyzing GTP to GDP and undergoing
conformational changes in the process. These factors are called
EF-Tu and EF-G in bacteria, and EF1 and EF2 in eukaryotes.
• Coupling the GTP hydrolysis-driven changes in the
elongation factors to transitions between different states of the
ribosome speeds up protein synthesis enormously.
• In addition to moving translation forward, EF-Tu increases its
accuracy
• First, the 16s rRNA in the small subunit of the ribosome
assesses the “correctness” of the codon–anticodon match by
folding around it and probing its molecular details
• When a correct match is found, the rRNA closes tightly around
the codon–anticodon pair, causing a conformational change in
the ribosome that triggers GTP hydrolysis by EF-Tu.
• Incorrect codon–anticodon matches do not readily trigger this
conformational change, and these errant tRNAs mostly fall off the ribosome before they can be
used in protein synthesis.
• There is a short time delay as the amino acid carried by the tRNA moves into position on the
ribosome. This time delay is shorter for correct than incorrect codon–anticodon pairs.
• An incorrect codon‒ anticodon interaction in the P site of the ribosome (which would occur after
the misincorporation) causes an increased rate of misreading in the A site. Successive rounds

of amino acid misincorporation eventually lead to premature
termination of the protein by release factors. The protein made will
therefore be degraded and never used.
In summary, there are two ways to ensure that right correct amino
acid is added:
1. Induced fit: folding of ribosome to ensure the correct geometry
2. Kinetic proofreading: GTP hydrolysis gives weaker interaction
between anticodons and tRNA; thus, the delay of getting into
position is much longer, causing the misreading and premature
ending of translation.
Ribosome Structure:
Ribosome is made of two thirds of RNA and one third of protein.
Ribosomal protein is mainly located in the surface to fill in the gaps
and crevices of the folded RNA. Ribosomal protein is mainly used to
stabilised the RNA core, while allowing the change in rRNA
conformation. It also aids in the initial assembly of the rRNAs that
make up the core of the ribosome.
Ribosome doesn’t contain easily ionisable function group that can be
used to catalyse chemical reaction nor metal ions. We find that 23s
rRNA forms a highly structured pocket that, through a network of
hydrogen bonds, precisely orient the two reactants and thereby
greatly accelerates their covalent joining.
Initiation of translation:
Eukaryotes:
• AUG is the starting codons of translation
• Initiator tRNA-methionine complex (Met-tRNAi) is first
loaded to small ribosomal unit along with the protein called
eukaryotic ignition factors (eIFs).
• small ribosomal unit binds to the 5’ end cap of the mRNA,
which is previous recognised by eIF4E and eIF4G
• small ribosomal unit then move forward to search for AUG
• Translation begins at the first encounter AUG. Initiation
complex dissociate, allowing large ribosomal unit to
assemble and complete the ribosome.
• Initiator tRNA remains at the P site, leaving A site empty.
• Leaky scanning: small ribosomal unit may skip the first
encounter AUG due to subtle difference from its recognition
sequence and jump to second AUG. It allows cell to produce
closely related protein from one mRNA.
Prokaryotes:
• Prokaryotic mRNA has no 5’ cap. So the
starting direction of translation can not
be found.
• Bacterial mRNA contains a specific
ribosome-binding sequence called
Shine-Dalgarno sequence, which
located few nucleotide upstream of
AUG.
• This nucleotide sequence forms a base

pair with 16s rRNA small ribosomal unit to position the
initialing AUG codon into the ribosome.
• There are several ribosome binding site in prokaryotic
mRNA. So material mRNA is polycistronic: they encode
several protein in one mRNA molecule.
Termination of translation:
• Stop codons signal the end of translation (UAA,UGA,UAG)
• Proteins known as release factors bind to any ribosome with a
stop codon positioned in the A site, forcing the peptidyl-
transferase in the ribosome to catalyze the addition of a water
molecule instead of an amino acid to the peptidyl-tRNA
• This reaction frees the carboxyl end of the growing polypeptide
chain from its attachment to a tRNA molecule, thereby releasing
the polypeptide chain
• Then the ribosome de-assemble into small and large subunits.
Polyribosomes:
• it is usual for multiple initiations to take place on each mRNA
molecule being translated. As soon as the preceding ribosome has
translated enough of the nucleotide sequence to move out of the 
way, the 5ʹ end of the mRNA is threaded into a new ribosome.
• These multiple initiations allow the cell to make many more protein
molecules in a given time than would be possible if each protein
had to be completed before the next could start.
• Because bacterial mRNA does not need to be processed and is
accessible to ribosomes while it is being made, ribosomes can
attach to the free end of a bacterial mRNA molecule and start
translating it even before the transcription of that RNA is
complete, following closely behind the RNA polymerase as it
moves along DNA.
Quality control to prevent translating damaged mRNA:
• To avoid translating broken mRNAs, for example, the 5ʹ cap and the poly-A tail are both
recognized by the translation-initiation machinery before translation begins
nonsense-mediated mRNA decay mechanism:

• As its 5ʹ end emerges from a nuclear pore, the mRNA is met by a ribosome, which begins to
translate it. As translation proceeds, the exon junction complexes (EJCs) that are bound to the
mRNA at each splice site are displaced by the moving ribosome. The normal stop codon will lie
within the last exon, so by the time the ribosome reaches it and stalls, no more EJCs will be
bound to the mRNA. In this case, the mRNA “passes inspection” and is released to the cytosol
where it can be translated.
• However, if the ribosome reaches a stop codon earlier, when EJCs remain bound, the mRNA
molecule is rapidly degraded. In this way, the first round of translation allows the cell to test the
fitness of each mRNA molecule as it exits the nucleus.
Folding of protein:
• Most proteins probably do not fold correctly during their synthesis and require a special class of
proteins called molecular chaperones to do so.
• Molecular chaperones are useful for cells because there are many different folding paths
available to an unfolded or partially folded protein.
• Molecular chaperones specifically recognize incorrect, off-pathway configurations by their
exposure of hydrophobic surfaces, which in correctly folded proteins are typically buried in the
interior.
• Chaperones prevent this from happening in normal proteins by binding to the exposed
hydrophobic surfaces using hydrophobic surfaces of their own.
• Many molecular chaperones are called heat-shock proteins (designated hsp), because they
are synthesized in dramatically increased amounts after a brief exposure of cells to an elevated
temperature . (42 degrees in 37 degrees of living cells) This reflects the operation of a feedback
system that responds to an increase in misfolded proteins.
• hsp70: acts early in the life of many proteins (often before the protein leaves the ribosome),
with each monomer of hsp70 binding to a string of about four or five hydrophobic amino
acids. On binding ATP, hsp70 releases the protein into solution allowing it a chance to re-fold.

• hsp60: form a large barrel-shaped structure that acts after a protein has been fully
synthesized.
• To enter a chamber, a substrate protein is ﬁrst captured via the hydrophobic entrance to the
chamber. The protein is then released into the interior of the chamber, which is lined with
hydrophilic surfaces, and the chamber is sealed with a lid, a step requiring ATP. Here, the
substrate is allowed to fold into its ﬁnal conformation in isolation, where there are no other
proteins with which to aggregate. When ATP is hydrolyzed, the lid pops open , and the
substrate protein, whether folded or not, is released from the chamber.
• The apparatus that deliberately destroys aberrant proteins is the proteasome, an abundant ATP-
dependent protease that constitutes nearly 1% of cell protein.
• Each proteasome consists of a central hollow cylinder (the 20S core proteasome) formed from
multiple protein subunits that assemble as a stack of four heptameric rings
• Each end of the cylinder is normally associated with a large protein complex (the 19S cap) that
contains a six-subunit protein ring through which target proteins are threaded into the
proteasome core, where they are degraded.
• The threading reaction, driven by ATP hydrolysis, unfolds the target proteins as they move
through the cap, exposing them to the proteases lining the proteasome core
• The proteins that make up the ring structure in the proteasome cap belong to a large class of
protein “unfoldases” known as AAA proteins.
• It must be able to distinguish abnormal proteins from those that are properly folded. The 19S cap
of the proteasome acts as a gate at the entrance to the inner proteolytic core, and only those
proteins marked for destruction are threaded through the cap.
• A special set of E3 molecules is responsible for the ubiquitylation of denatured or otherwise
misfolded proteins, as well as proteins containing oxidized or other abnormal amino acids.
Abnormal proteins tend to display on their surface hydrophobic amino acid sequences or
conformational motifs that are recognized as degradation signals by these E3 molecules; these
sequences are buried and therefore inaccessible in the normal, properly folded version.

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