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Dudi Engelberg
Room 1-517
Tel: 658 4718
e-mail: [email protected]
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The central dogma
of molecular biology
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DNA
Transcription
RNA
Translation
Protein
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Could proteins multiply ?
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What do we have RNA for?
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Same DNA content in all cells of the
mulicellular organism?
What is the function of DNA?
Can cells function without DNA?
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Are these all nucleotides that appear in DNA and RNA?
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What are the cellular functions of nucleotides?
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Some cellular functions of nucleotides
1. Building blocks of nucleic acids.
2. Energy carrier (ATP, GTP).
3. Building parts of enzymes co-factors (e.g., NAD, FAD,
CoenzymeA, S-adenosylmethionine).
4. Regulators in signal transduction processes.
5. Second messengers in signal transduction (cAMP, cGMP).
6. Phosphate donors in phosphorylation reactions. Involved in many
more pottranslational modifications.
7. Serve as structural molecules (rRNA).
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8. Activators of carbohydrates for synthesis (glycogen for example).
Some cellular functions of deoxynucleotides
1. Building blocks of nucleic acids (DNA).
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Some cellular functions of deoxynucleotides
1. Building blocks of nucleic acids (DNA).
2. Energy carrier (ATP, GTP).
3. Building parts of enzymes co-factors (e.g., NAD, FAD,
CoenzymeA, S-adenosylmethionine).
4. Regulators in signal transduction processes (GTP).
5. Second messengers in signal transduction (cAMP, cGMP).
6. Phosphate donors in phosphorylation reactions.
7. Serve as structural molecules (rRNA).
8. Activators of carbohydrates for synthesis (glycogen for example).
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Some deviations from the averaged Watson & Crick model
The pitch angle between base pairs could be 28o - 42o.
Bases could propel (deviate from planarity).
Damages: kinks and covalent bonding inside the helix (usually
Between bases).
Presence of unusual bases (in tRNA for example) allows unusual
base pairing and novel structural motifs.
Presence of specific sequences (stretch of purines,
palindromes, sequence repeats).
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The driving force towards
synthesis is the breakdown of
PPi.
Phosphodiester bond
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Mechanism of the basic synthesis reaction of
nucleic acids
Addition of nucleotide involves an attack by the 3’-hydroxyl group
at the end of the growing RNA molecule on the a phosphate of the
oncoming NTP.
Two Mg2+ ions coordinated to the phosphate groups of the NTP and
to three Asp residues of the  subunit of E. coli RNA polymerase
(conserved in most RNA polymerasess in nature).
One Mg2+ ion facilitate the attack by the 3’-hydroxyl group on the a
phosphate and the other ion facilitates the displacement of
pyrophosphate.
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The Mg2+ ions stabilize in fact the transition (intermediate) state.
Polymerization of nucleotides - DNA and RNA biosynthesis
1. The reaction is directional; proceeds from 5’end to 3’end.
As a result the product is asymetric (5’end different than
3’end.
2. The nucleotides (of the same strand) are always linked
in a phospho-di-ester bond (a covalent bond).
3. Energy is wasted in addition of each monomer. The
driving force towards synthesis is degradation of
pyrophosphate.
4. The precursors are always nucleotides tri-phosphates
(NTPs or dNTPs).
6. The reaction is directed by a pre exist plan (a template).
(No polymerase is capable of adding nucleotides randomly).
May be there are some - quite important
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Basic characteristics of DNA Pol
1. Is not capable of de novo synthesis.
Requires:
A. A template (as any other polymerase).
B. A primer (RNA oligo, nicked DNA, protein?)
2. Possesses two catalytic activities:
A. A 5’ to 3’ polymerase activity.
B. A 3’ to 5’ exonuclease actiivty.
3. Substrates are only dNTPs.
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How DNA Pol is regulated?
Does it possess regulatory
sites?
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DNA replication is semi-conservative
DNA replication is bi-directional
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Schematic structure of E. coli replication origin (OriC)
245 bp.
3 repeats of 13 bp sequences + 4 repeats of 9 bp sequence.
These elements are highly conserved in replicationorigins
of bacteria.
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Initiation step: “opening” DNA “preparing the template before
any DNA synthesis occurs.
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First key step in replication: binding of DnaA protein molecules
to the four 9 bp repeats.
DnaA binding requires ATP and HU
Second step: binding of DnaB (hexamerix helicase). Two hexamers bind to unwind
DNA at two points creating two potential replicating forks.
Third step: binding of SSBs (essential for stabilizing single strand throughout the
replication process) and DNA gyrase (DNA topoII) - this step allows DnaB
helicase to unwind thousands of base-pairs.
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DnaA binds cooperatively to form a core
around which OriC DNA is wrapped.
At the presence of ATP DnaA melts the
DNA of the A-T rich 13 bp tandem repeats.
DnaA molecules recruit two DnaB-DnaC
complexes, one for each replication forks.
(6 DnaC monomers bind the DnaB hexamer.)
Gyrase must be present to relieve topological
Stress - otherwise helicase cannot further
catalyze unwinding.
Altogether a pre-priming complex is formed:
480 kD, 6 nm radius.
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Initiation step has prepared the template.
Moving to elongation step:
Priming is required.
A mechanism for bi-directionality is required.
Leading strand synthesis begins with
The synthesis of a short primer (10-60 n)
catalyzed by primase (DnaG - special
RNA Pol).
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Both strands are sybthesized by DNA Pol3.
Lagging strand:
A new primer is synthesized near
the replication fork.
Synthesis continues until the
Fragment extends as far as the primer
of the previous fragment.
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Specific structural capabilities of
DNA Pol 3.
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DnaB (helicase) + DnaG (primase) form a functional unit
within the replication fork, called primosome.
DNA pol3 - a dimer - one set of subunits synthesize the
leading strand and other set the lagging strand.
Once DNA is unwound by DnaB, DnaG associates occasionally
with DnaB and synthesizes a short RNA primer.
A new  sliding clamp is then positioned at the primer by the
clamp-loading complex of Pol 3.
When a synthesis of a fragment is completed, replication
halts and the core subunits of Pol 3 dissociate from their 
sliding clamp and from the new fragment.
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 subunits on DNA
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Exonuclease activity is located
ahead of pol activity
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Sequence of the RNA is identical to that of the coding strand
(with the replacements of Us for Ts).
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Products of the transcription reaction (primary transcript):
In prokaryotes: an unstable RNA- rapidly degraded (mRNA
or cleaved to give mature products (rRNA, tRNA).
In eukaryotes: modified at the ends (mRNA) and/or cleaved
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to give mature products (all RNAs).
With the exception of the RNA genomes
of certain viruses, all RNA molecules in
nature (mRNA, tRNA, rRNA, miRNA,
snRNA) are derived from information
stored in DNA and obtained
via transcription.
Namely, just like DNA during replication,
RNA is synthesized on DNA
template (DNA-dependent RNA synthesis).
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Transcription=DNA-dependent RNA synthesis
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Polymerization of nucleotides - DNA and RNA biosynthesis
1. The reaction is directional; proceeds from 5’end to 3’end.
As a result the product is asymetric (5’end different than
3’end.
2. The nucleotides (of the same strand) are always linked
in a phospho-di-ester bond (a saturated covalent bond).
3. Energy is consumed during addition of each monomer. The
driving force towards synthesis is degradation of
pyrophosphate.
4. The precursors are always nucleotides tri-phosphates
(NTPs or dNTPs).
6. The reaction is directed by a pre exist plan (a template).
No plymerase is capable of adding nucleotides randomly.
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At its basic enzymatic level, transcription is a
reaction highly similar to replication
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Comparison of replication to transcription (some aspects)
Replication
Quantity:
The whole genome
Transcription
Parts of the genome
Timing:
One time per life cycle
(time is determined by the
checkpoint system)
some parts - all life time
some parts - some time
some parts - never
Location:
From origin to end
Many starts and many stops
(starts and stops must be
most accurate)
DNA substrate:
The two strands
One strand (could be a
different for each particular
case
Nucleotide
substrates:
dNTPs
NTPs
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Comparison of replication to transcription (some aspects)
Replication
Proofreading:
Post-reaction
repair:
Fate of
product:
Always
Always
Remains attached to
template
Processivity:
High or low
Ligating
fragments:
Yes
Transcription
Never
Never
Released from
template
High (from start to
termination)
No - products are
independent molecules
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Sequence of the RNA is identical to that of the coding strand
(with the replacements of Us for Ts).
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Products of the transcription reaction (primary transcript):
In prokaryotes: an unstable RNA- rapidly degraded (mRNA
or cleaved to give mature products (rRNA, tRNA).
In eukaryotes: modified at the ends (mRNA) and/or cleaved
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to give mature products (all RNAs).
RNA Polymerase - general properties
1. Properties similar to DNA Polymerases:
- Basic chemical mechanisms: addition of ribonucleotides to the 3’-OH of the
chain. Consequently determination of a 5’ to 3’ directionality.
- Requires a template.
- Adding nucleotides on the basis of optimal hydrogen bonds with the template
strand (A-U, C-G).
2. Properties specific to RNA Pol
- Using only one strand as a template (must make a choice).
- Does not require a primer (pppN 5’ end).
- Very complex regulation for “choosing” the starting points (which may be
different in every cell, in every developmental stage and in ageing.
- Does not have a 3’
5’ exonuclease activity.
- The rate of mistake in high (1/104-105).
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During a successful round: RNA Pol associates with the starting point
and dissociates at the termination point, defining a transcription
unit. A transcription unit may include more than one gene
Nomenclature:
Upstream.
Downstream; numbers;
left to right; no base is
defined as base zero.
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Rates (in E. coli):
Transcription: 40 nuc../sec.
Similar to rate of translation.
Replication: 1,000nuc./sec/strand
RNA pol creates the
‘transcription bubble’ when
It binds to a DNA. The bubble
moves with it.
Displacing of the product
(RNA),
reforming the dsDNA
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About 17 bp are unwound at any given time.
Length of RNA:DNA hybrid within the bubble: up to 12 bp.
Length of RNA within the bubble: ~25 b.
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Within the transcriptional bubble (in bacteria), RNA Pol :
Unwinds and rewinds DNA
Maintains the conditions of the template and coding strands.
Synthesizes RNA.
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The transcription reaction can be divided into the
Following stages:
Template recognition - binding of RNA pol to DNA
at a sequence known as promoter forming a “closed
complex”, unwind the DNA to form an “open complex”,
creating the ‘bubble’.
Initiation - synthesis of the first nucleotide bond. RNA pol
Does not move while it synthesizes the first ~9 bases.
Abortive events may occur, forcing initiation to start again.
Initiation phase ends when the enzyme succeeds in extending
the chain and clears the promoter.
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Elongation - enzymes moves along the DNA, extending the
RNA, unwinding the DNA exposing new segments of the
template and displace the RNA-DNA hybrid to re-form
the original double stranded DNA. RNA emerges as a free
single strand.
Termination - recognition of the point at which no further
bases should be added to the chain. The enzyme and the
RNA should be released and the DNA re-forms the original
duplex state.
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Initiation of transcription: a crucial
(some time the only) regulatory
step in gene expression.
Some key questions:
How starting point is recognized?
How initiation rate is determined?
The transcription bubble: transiently
and shortly separation of the DNA
to single strands.
The process of transcription: the usual
complementary base pairing process.
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Stages in which the bubble is created
Template recognition. Closed complex.
Local unwinding: open complex
(template strand is available)
Initiation (up to 9 bases that could be
released; no move)
Promoter clearance
Elongation - Movement of the bubble.
(inchworm move or fluent?)
Termination:1. Cease addition of nucleotides.
2. Set complex apart.
Just like initiation, termination is
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sequence-dependent. Defines the terminator.
Promoter: The sequence of DNA needed for RNA
polymerase to bind to the template and accomplish
the initiation reaction (synthesis of the first nucleotide
bonds).
Terminator: The sequence of DNA required for
disrupting the bubble and reforming the DNA duplex
(after the last base is added).
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 an ’ subunits have
a channel for the DNA
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Yeast RNA Pol II is composed of 12 subunits (holoenzyme). Two
subunits form a different sub-complex. Two subunits are not essential
for
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viability.
Following DNA binding and melting, the
“clamp” swings back to force a turn. [note, colors of subunits82are
the same as in the crystal structure]
“wall” protein is enforcing
a turn.
The length of RNA hybrid
is limited by the activity of
the “rudder” protein. The
RNA is forced to leave the DNA
When it hits the protein rudder.
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The bridge protein is found in different conformations
In different crystal structures.
Probably, breaking and re-making of contacts
is mediated by conformational changes
of the “bridge” protein:
A nucleotide addition cycle:
1. The bridge is in a straight conformation adjacent to
the nucleotide entry site.
2. After adding a nucleotide to the RNA the bridge
protein is in contacts with the newly added nucleotide,
undergoes a conformational change and moves one base
pair along the template, obscuring the nucleotide entry
site.
3. The bridge returns to its straight conformation, allowing
Entry of next nucleotide of the template - namely,
the bridge acts as a ratchet.
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Properties of the core enzyme
The core enzyme of E. coli has a general affinity for DNA (driven
by electrostatic attraction between the basic protein and
the acidic DNA). Yet, it does not distinguish between promoters and
other sequences.
Any random sequence bound by core enzyme is described
as a “loose binding site”. No change occurs in the DNA
which remains duplex.
Such a core enzyme-DNA complex is stable (half life for
dissociation is 60 min.).
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Properties of the holoenzyme
The holoenzyme has a
drastically reduced ability
to recognize “loose binding
sites” (half life of <1sec. Kd
reduced by a factor of 104).
The holoenzyme binds
promoters with Kds 1,000
time higher than core
enzyme with half lives of
hours.
However, it manifests a
specific Kd to any specific
promoter.
Sigma confers the ability to
recognize specific sites. It is
also involved in “melting”,
creating an “open” complex.
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Depending of specific promoter the Kd for DNA:RNA pol
association is 106 - 1012 (first level of regulation of rate of
transcription).
Formation of an open complex by melting (that is driven by
sigma) allows tight binding that is not reversible.
Initiation rate (frequency of initiation) also differs
(dependent on other factors in addition to RNA pol:DNA
associatio. Frequencies can range between 1/sec (rRNA genes
to 1/30 min. (lacI promoter).
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The holoenzyme binds
promoters with Kds 1,000
time higher than core
enzyme with half lives of
hours.
This property assists with promoter
recognition, but significantly interferes
with elongation. Therefore, sigma dissociates
from the enzyme when elongation starts.
Sigma factor is recycled.
It becomes unnecessary when
abortive initiation is concluded.
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(sigma)
(promoter
region)
Sigma contacts mainly bases of the coding strand and continues to
hold these contacts - an important step in melting (forming an “open
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complex and recognition of template strand.
What is responsible for the ability of holoenzyme to bind
specifically to promoters?
Sigma has domains that recognize promoter DNA, but as an independent protein
Sigma does not bind to DNA. There is major change in conformation of sigma
when it binds core enzyme. The N-terminal region of free sigma suppresses the
activity of the DNA-binding region - it is an autoinhibitory domain.
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How holoenzyme finds a specific promoter (60bp in a 4x106 stretch)?
The forward rate constant for RNA Pol binding to promoters is faster than
random diffusion (that limits the constant to 108/M-1Sec-1).
The measured rate constant for association with a 60 bp target
is 1014/M-1Sec-1.
If the target is the whole genome the rate constant is around 1014/M-1Sec-1.
But how does the polymerase move from random binding sites to promoters?
Perhaps RNA Pol binds DNA and remains contact
(no simple diffusion that relies on random binding). Rather, a direct
Displacement with other sequence occurs (no sliding).
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The “diffusion model: random association with loose sites on DNA,
dissociation and re-bind, until occasionally (statistically) interacting
with a promoter, and remains associated.
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A direct displacement
model - diffusion is not
required
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Promoter’s function is provided directly by
its DNA sequence/structure (it does not need to be
transcribed or translated).
It is a cis-acting site.
[in genetic terminology, sites that are located on the
same DNA are said to be in cis. Sites that are located
on two different molecules of DNA are being in
trans.]
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Conserved - a base most often present at a position.
Perhaps the most striking feature of E. coli promoters is the lack of extensive conservation
of sequence over the 60 bp associated with RNA Pol.
Promoter elements (in E. coli):
Start point (a purine in 90% of the RNAs).
-10 sequence
-35 sequence
The distance separating the -35 and the -10 sites.
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The -10 sequence:
T80A95T45A60A50T96
Sequence that resides in poistions of -18 to -9 in all known
E. coli promoters.
Subscripts denote the percent occurrence of the most frequent found
base
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The -35 sequence:
T82T84G78A65C54A45
The distance separating the -35 and -10 sites is between 16-18 bp
in 90% of promoters. In the exceptions it can go down to 15 or up
to 20. Sequence itself is not important.
Some promoters have an A-T-rich sequence located farther upstream.
It is called UP element and interacts with a subunit of RNA pol. It
Is typically found in promoters that are highly expressed, such as the
promoters of the rRNA genes.
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Up elements are associated with a subunit of RNA pol. Found in promoters that
are highly expressed.
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In spite of conservation of promoters there is ~1000 fold variation
in the rate at which RNA polymerase binds to different promoters
in vitro.
Binding rates correlate well with the frequencies of transcription
in vivo.
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Sequences at prokaryotic terminators
show no similarities.
Many terminators require a hairpin to
form.
Termination involves recognition of
signals on the transcript.
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Hairpin structure
+ U rich sequence
(1100 sequences in
E. Coli fit these criteria.
Intrinsic terminator - other factors
are not required. Works in vitro
too.
Hairpins may cause polymerase
to slow or even to stop.
Antitermination process may
allow RNA Pol to continue
(readthrough).
Downstream U-rich destabilizes
RNA-DNA hybrid.
Hairpin + U-rich are
Necessary, but not sufficient.
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The weakest base-pair is
the rU-dA
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Rho:
A 275 Kd homo-hexamer.
RNA binding domain + ATPase domain.
Belong to a family of ATP-dependent helicases.
Functions as an ancillary factor for RNA Pol.
Most efficient at 10% concentration.
Accounts for about 50% of terminations in E. coli.
Rho-dependent termination sequences are rich in
Cs and poor in Gs. Reside 50-90 bases from
termination sites.
Acts processively on a single RNA substrate.
Moves faster than RNA Pol.
Pausing is important for Rho-dependent termination
too.
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Translation
Components involved in translation account for 35%
of the dry weight of E. coli cells.
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A condensation reaction: formation of the peptide bond by removal of
water (dehydration) from the -carboxyl group of one amino acid and
the -amino group of another
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To make the reaction thermodynamically more
favorable, the carboxyl group must be
chemically modified or activated so that the
hydroxyl group can be more readily eliminated
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(Dihydrouridine)
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First stage in translation: aminoacyl-tRNA synthetases esterify
the 20 amino acids to their corresponding tRNA.
Each enzyme is specific for one amino acid and one or more tRNAs.
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Step 1: An enzyme-bound intermediate
Aminoacyl-AMP forms when the carboxyl
group of the amino acid reacts with the
-phosphoryl group of ATP, creating an
anhydride linkage, with displacement of
pyrphosphate.
Step 2: The aminoacyl group is
transferred to its corresponding tRNA.
The resulting ester linkage has a highly
negative standard free energy of
hydrolysis.
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Valine and isoleucine differ in only a single methylene group
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Proofreading by aminoacyl-tRNA synthetases
Two active sites in the Ile-tRNAIle synthetase:
- binding of the amino acid to the enzyme (affinity to
Ile is only a little higher than affinity to Val (error in
1/200 entries.
- binding of aminoacyl-AMP product. This site has higher
affinity to AMP-Val. A hydrolytic site.
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What is accomplished by aminoacylation of tRNA?
1. Activation of the amino acid for peptide bond formation.
2. Attachment of the amino acid to an adaptor tRNA that ensures
appropriate placement of the amino acid in a growing polypeptide.
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N-formyl group is added to the
amino group of methionine by
transformylase.
Transformylase is specific to
Met attached to tRNAfMet
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Translation initiation in prokaryotes
IF-3 prevents combining of the 30S
and 50S subunits
The initiating 5’AUG is guided to its correct
position by the Shine-Delgarno sequence
in the 5’UTR of the mRNA (AUG is the
beginning of an ‘open reading frame’).
The initiating 5’AUG is positioned at a site
called the P site, the only site in the ribosome
to which fMet-tRNAfMet can bind.
The fMet-tRNAfMet is the only aminoacyl-tRNA
that binds first to the P site.
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Step 2 in translation initiation:
GTP-bound IF-2 and the fMet-tRNAfMet join the ribosome, guided
by the anticodon that pairs with the mRNA initiation codon.
Step 3 in translation initiation:
The complex (30S + IF1,IF2-GTP,IF3 + fMet-tRNAfMet) combines with
the 50S ribosomal subunit; simultaneously, the GTP bound to
IF-2 is hydrolyzed to GDP and Pi which are released from the
complex. All 3 initiation factors are also released from the complex.
IF-2-GDP is re-loaded with GTP via a GDP/GTP exchange reaction.
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Translational elongation
Step 1:
Appropriate incoming aminoacyl-tRNA
binds to a complex of GTP bound EF-Tu.
The GTP-EF-Tu-aminacyl-tRNA complex
binds the A site of the 70S complex.
The GTP is hydrolyzed and the EF-Tu-GDP
is released.
EF-Tu-GTP complex is regenerated via a
GDP/GTP exchange reaction catalyzed
by EF-Ts.
AA2
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Translation elongation, Step 2: Formation of the peptide bond:
The -amino group of the amino
acid in the A site acts as a
nucleophile, displacing the tRNA
in the P site to form a peptide bond.
The tRNAfMet at the P site is now
uncharged.
The peptidyl transferase reaction
is probably catalyzed by the
23S rRNA
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Translation elongation, Step 3: Translocation
Move of the ribosome. The ribosome
moves one codon towards the 3’ end of
the mRNA.
Translocation is catalyzed
by EF-G-GTP (translocase).
The ribosome is now ready for the
next elongation cycle.
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Termination
Catalyzed by RF1 or RF2.
(depending on the particular stop codon).
RF1 and RF2 are proposed to mimic the
structure of tRNA.
RF-1 recognizes UAG and UAA. RF-2
Recognizes UGA and UAA.
In eukaryotes, a single RF, eRF, recognizes
all 3 termination codons.
Releasing factors:
1. Hydrolyze the ester linkage of the
peptydil-tRNA bond.
2. Release the polypeptide and the last
tRNA (now uncharged).
3. Dissociate the ribosome to 30S and 50S
subunits.
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EF-Tu
EF-G
The carboxy terminal domain of EF-G mimics the structure of tRNA.
Altogether EF-G mimics the structure of EF-Tu-tRNA complex
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probably binds to the A site and displacing the peptidyl-tRNA.
Translation is energy consuming:
On average, hydrolysis of more than 4 NTPs to NDPs is
required for the formation of each peptide bond of a polypeptide.
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Bacterial ribosome’s M.W.:
~2.7 million
Components in the ribosome structure:
Proteins: blue (in large subunit); Yellow (in small subunit).
Bases of rRNA in large subunit: white. Backbone of rRNA in large
subunit: green. rRNA in small subunit: white. tRNAs: purpule,
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mauve, gray. mRNA contacting tRNAs: red.
In the 50S subunit, the 5S and 23S rRNAs form the structural core.
The proteins are secondary elements in the complex, decorating the
surface.
No protein is detected within 18A of the active site for peptide
bond formation.
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50S subunit of a bacterial
ribosome.
Red - a puromycine molecule
at the active
peptidyl transferase site. Note
no proteins in the vicinity.
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Steady state level of a protein (expression level) is determined
by a combination of regulation of:
Transcription initiation
mRNA degradation (mRNA stability)
mRNA processing
Transport to cytoplasm
Translational control
Folding and protein processing
Protein degradation (protein stability)
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