Chapter 6 Summary

Molecular Biology and Cell Biology

RNA: linear, single-stranded polymer of ribonucleotides (A, U, G, C)
linked together by phosphodiester bonds.
Transcription of DNA differs from replication:
The formed RNA strand does not remain hydrogen-bonded to
the DNA template strand, but rather it is displaced and the
DNA helix reforms.
RNA molecules are released as a single strand.
They are copied from only a limited region of NDA much
shorter.
RNA is single stranded, but contains
short stretched of nucleotides that can
form conventional base pairs with
complementary sequences allows RNA
to fold into a 3D structure.
RNA polymerases: enzymes that
perform transcription catalyze the
formation of the phosphodiester bonds
that link the nucleotides together. The
RNA chain is extended one nucleotide
at a time in the 5 to 3 direction. The
substrates are ribonucleoside
triphosphates (ATP, CTP, UTP, GTP), the
hydrolysis of which provides the energy
needed for the reaction.
Differences RNA polymerase and DNA polymerase:
RNA polymerase catalyzes the linkage of ribonucleotides, not
deoxyribonucleotides.
RNA polymerases can start an RNA chain without a primer.
RNA polymerases are completely processive they do not
make their products in segments but one RNA polymerase
makes the whole RNA polymer.

The
RNA

molecules that are copied from the genes that specify amino acid
sequences of proteins are called messenger RNA (mRNA). The
final product of other genes is the RNA itself, known as non-coding
RNA serve as structural, enzymatic and regulatory components of
cell processes.
Transcription unit: each transcribed segment of DNA.
^ The transcription cycle of bacterial RNA polymerase.

1. The RNA polymerase holoenzyme (polymerase + sigma factor)

assembles and locates a promoter DNA sequence.
2. It unwinds the DNA at the position at which transcription is to
begin.
3. Transcription begins. The initial RNA synthesis (abortive
initiation) is relatively inefficient as short, unproductive
transcripts are released.
4. Once RNA polymerase has synthesizes about 10 nucleotides
of RNA, it breaks its interactions with the promoter DNA and
releases the sigma factor.
5. The polymerase tightens around the DNA and shifts to the
elongation mode for RNA synthesis, moving along the DNA.
6. During elongation mode, transcription is highly processive.
7. The polymerase leaves the DNA template and releases the
newly transcribed RNA only when it encounters a termination
signal, which is typically encoded in DNA.

Termination signals consist of a string of A-T nucleotide pairs

preceded by a twofold symmetric DNA sequence, which when
transcribed into RNA folds into a hairpin structure through Watsoncrick base pairing and helps to disengage the RNA transcript from
the active site.
A consensus nucleotide sequence is derived by comparing many
sequences with the same basic function (eg. promoter) and tallying
up the most common nucleotides found at each position gives an
average. A sequence logo shows the relative frequencies of each
nucleotide at each position.
Eukaryotes have 3 types of RNA polymerases. I and III transcribe the
genes encoding transfer DNA, ribosomal DNA and various small
RNAs. II transcribes most genes. Differences with bacterial RNA
polymerase:

1. Eukaryotic RNA polymerases require

many transcription initiation factors (such
as the sigma factor) called general
transcription factors.
2. Eukaryotic transcription initiation must
take place on DNA thats packaged into
nucleosomes and higher-order forms or
chromatin structure, which dont exist in
bacterial chromosomes.
Initiation of transcription of a eukaryotic gene
by RNA polymerase II:
1. The promoter contains a DNA sequence
called the TATA box, which is located 25
nucleotides away from the site at which
transcription is initiated.
2. Through its subunit TBP, TFIID recognizes
and binds the TATA box, which enables
the adjacent binding of TFIIB.
3. The rest of the general transcription
factors and the polymerase assemble at
the promoter to form a transcription
initiation complex.
4. TFIIH uses energy from ATP hydrolysis to
pry apart the DNA helix at the
transcription start point, and
phosphorylates RNA polymerase II so that
the general factors are released and it
can begin elongation.
5. The site of phosphorylation is a long Cterminal polypeptide tail (C-terminal
domain, CTD).
Transcription initiation in vivo requires the
presence of transcription activator proteins,
which bind to specific short sequences in DNA and determine rate
and pattern of transcription. Help all the parts to assemble at the
promoter, and then attract ATP=dependent chromatin remodeling
complexes and histone-modifying enzymes.

If rotation is
prevented,
superhelical tension
formed by the
opening of
nucleotide pairs is
introduced to the
DNA strand. To
relieve the tension,
the DNA helix bends
into supercoiled
loops. It forms one
supercoil for every
10 nucleotide pairs
opened.

Comparisons of the steps

leading from gene to protein in
eukaryotes and prokaryotes.
In eukaryotes, DNA
topoisomerase enzymes rapidly
remove the superhelical tension caused by proteins propelling

themselves along the DNA strand. In bacteria, DNA gyrase uses the
energy of ATP hydrolysis to pump supercoils continuously into the
DNA, thereby maintaining the DNA under constant tension. These
are negative supercoils, having the opposite handedness from
positive supercoils that form when a region of DNA helix opens it
undoes its effects, thereby making the opening of DNA helix in this
region energetically favorable compared with DNA helix that is not
yet supercoiled.
Both ends of
eukaryotic mRNAs are
modified: capping on
the 5 end and
polyadenylation of the
3 end, which allow the
cell to assess whether
both ends of an mRNA
molecule are present
(and message is
intact) before the RNA
is exported from the
nucleus.
Bacterial mRNAs can
contain the
instructions for several
different proteins,
whereas eukaryotic
mRNA contains the info for a single protein.

RNA capping occurs after the first

25 nucleotides. A cap consists of a
modified guanine nucleotide.
1. A phosphatase removes a
phosphate from the 5-end of the nascent RNA.
2. A guanyl transferase adds a GMP in reverse linkage (5 to 5
instead of 5 to 3)
3. A methyl transferase adds a methyl group to the guanosine.
The 5 methyl cap signifies the 5 end of eukaryotic mRNAs, and this
helps to distinguish mRNAs from other RNA types. RNA polymerase I
and III produce uncapped RNAs during transcription, as they lack a
CTD.

In the pre-mRNA splicing reaction (left),

a specific adenine nucleotide in the
intron sequence (red) attacks the 5
splice site and cuts the sugar-phosphate
bond backbone of the RNA at this point.
The cut end becomes covalently bonded
to the adenine nucleotide, creating a
loop. The released free 3 OH end of the
exon sequence reacts with the start of
the next exon sequence, joining them
together.

There are two types of splicing errors:

1. Exon skipping
2. Cryptic splicing. Cryptic splicing
signals are nucleotides sequences
of RNA that closely resemble true
splicing signals and are
sometimes mistakenly used by the
spliceosome.

A
strategy
called
exon

definition helps to choose the appropriate splice sites. Exon size

tends to be more uniform than intron size. Through exon definition,
the splicing machinery can seek out the relatively homogenously
sized exon sequences.
To the left are the major steps in generating the 3 end of a
eukaryotic mRNA.

Only when proteins present on an

mRNA molecule collectively signify
that processing was successfully
completed, is it exported from the
nucleus. Improperly processed
mRNAs and other RNA debris
(introns) are retained in the nucleus,
where they are eventually degraded
by the nuclear exosome.
Successfully processed mRNAs are
guided through the nuclear pore
complexes (NPCs) aqueous
channels in the nuclear membrane
that directly connect the
nucleoplasm and cytosol.
Macromolecules are moved through
by nuclear transport receptors. A
specific one must be loaded onto the
mRNA, which takes place in concert
with the 3 cleavage and
polyadenylation.
Of all the proteins that assemble on
pre-mRNA molecules as they emerge
from transcribing RNA polymerases,
the most abundant are the hnRNPs
(heterogeneous nuclear ribonuclear
proteins). Some unwind the hairpin
helices in the RNA so splicing and
other signals on the RNA can be read
more easily. Others preferentially
package the RNA contained in the
very long intron sequences.

mRNA production is made more efficient in the nucleus by an

aggregation of the many components needed for transcription and
pre-mRNA processing. A postulated scaffold protein holds various
components in the proximity of a transcribing RNA polymerase.
Other key components are bound directly to the RNA polymerase
tail. A large number of such scaffolds are brought together to form
an aggregate that is highly enriched in the many components
needed for the synthesis and processing of pre-mRNAs.
Codons are always written with the 5-terminal nucleotide to the left.
The translation of mRNA into protein
depends on adaptor molecules that
can recognize and bind both to the
codon and, at another site, to the
amino acid. These adaptors consist
of transfer RNAs (tRNA).

Like most other eukaryotic RNAs,

tRNAs are covalently modified before
they are allowed to exit from the
nucleus. They are synthesized at larger
precursor tRNAs, which are then
trimmed. Some also contains introns
that must be spliced out, using a cutand-paste method catalyzed by
proteins. Because misfolded tRNA
precursors will not be processed
properly, the trimming and splicing
reactions serve as quality-control steps
in the generation of tRNAs.
All tRNAs are modified chemically
nearly 1 in 10 nucleotides is an altered
version of G U C or A ribonucleotides.
Some modified nucleotides, especially
inosine (produced by the deamination
of adenosine) affect the conformation
and base pairing of the anticodon and
thereby facilitate recognition of the
appropriate mRNA codon by the tRNA
molecule.
An amino acid is activated for protein
synthesis by an aminoacyl-tRNA
synthetase enzyme in 2 steps. The
energy of ATP hydrolysis is used to
attach each amino acid to its tRNA
molecule in a high-energy linkage. The
amino acid is first activated through
the linkage of its carboxyl group
directly to AMO, forming an adenylated amino acid. Without leaving
the synthase enzyme, the AMP-linked carboxyl group on the amino
acid is then transferred to a hydroxyl group on the sugar at the 3
end of the tRNA molecule, which joins the amino acid by an
activated ester linkage to the tRNA and forms the final aminoacyltRNA molecule.

The structure of the aminoacyl-tRNA linkage the carboxyl end of

the amino acid forms an ester bond to ribose. Because the
hydrolysis of this ester bond is associated with a large favorable
change in free energy, an amino acid held in this way is said to be
activated.

The genetic code is translated by means of two adaptors that act

one after another. The first adaptor is the aminoacyl-tRNA
synthetase, which couples a particular amino acid to its
corresponding tRNA. The second adaptor is the tRNA molecule itself,
whose anticodon forms base pairs with the appropriate codon on the
mRNA. An error in either step would cause the wrong amino acid to
be incorporated into a protein chain.
With hydrolytic editing, which occurs after the amino acid has been
covalently linked to AMP, the synthetase tries to force the
adenylated amino acid into a second editing pocket in the enzyme
when tRNA binds. The precise dimensions of this pocket exclude the

correct amino acid, while allowing access by closely related amino

acids. In the editing pocket, an amino acid is removed from the AMP

by hydrolysis. This increases the overall accuracy of tRNA charging

to 1 mistake in 40.000 couplings.
A polypeptide chain grows by the stepwise addition of amino acids
to its C-terminal end. The formation of each peptide bond is
energetically favorable because the growing C-terminus has been
activated by the covalent attachment of a tRNA molecule. The
peptidyl-tRNA linkage that activates the growing end is regenerated
during each addition.

The synthesis of proteins is guided by

information carried by mRNA molecules.
To maintain the correct reading frame
and to ensure accuracy, protein
synthesis is performed in the
ribosome, a complex catalytic machine
made from more than 50 proteins and several RNA molecules
(ribosomal RNAs, rRNAs). The large and small ribosome subunits are
assembled at the nucleolus, where newly transcribed and modified
rRNAs associate with the ribosomal proteins that have been
transported into the nucleus after their synthesis in the cytoplasm.
These two ribosomal subunits are then exported to the cytoplasm,
where they join together to synthesize proteins.
Each ribosome has one binding site for mRNA and three binding
sites for tRNA: the A (aminoacyl-tRNA), P (peptidyl-tRNA) and E
(exit) sites.
A tRNA molecule is held tightly at the A and P sites only if its
anticodon forms base pairs with a complementary codon (allowing
for wobble) on the mRNA molecule that is threaded though the
ribosome. The A and P sites are close enough together for their two
tRNA molecules to be forced to form base pairs with adjacent
codons on the mRNA molecule, which maintains the correct reading
frame on the mRNA.

< Translating an mRNA molecule. Each

amino acid added to the growing end of
a polypeptide chain is selected by
complementary base pairing between
the anticodon on its attached tRNA
molecule and the next codon on the
mRNA chain. Because only one of the
many types of tRNA in a cell can base
pair with each codon, the codon
determines the specific amino acid to be
added to the growing polypeptide chain.
The four-step cycle is repeated over and
over during protein synthesis.
2. A new peptide bond is formed.
3. The large subunit translocates
relative to the small subunit,
leaving the two tRNAs in hybrid
sites: P on the large subunit and A
on the small, for the small.
4. The small subunit translocates
carrying its mRNA a distance of 3
nucleotides through the ribosome.
This resets the ribosome with a
fully empty A site, ready for the
next aminoacyl-tRNA molecule to
bind.
mRNA is translated in the 5-to-3
direction, and the N-terminal end of a
protein is made first, with each cycle
adding one amino acid to the Cterminus of the polypeptide chain.

< Expansion of the previous figure, showing a

detailed view of the translation cycle. EF-Tu
and EF-G drive translation in the forward
direction. EF-Tu provides opportunities for
proofreading of the codon-anticodon match,
so incorrectly paired tRNAs are selectively
rejected. The binding of a molecule of EF-G to
the ribosome and the subsequent hydrolysis
of GTP lead to a rearrangement of the
ribosome structure, moving the mRNA being
decoded exactly 3 nucleotides through it.

 The initiation of protein synthesis in

eukaryotes. Only three of the many
translation initiation factors (eIFs) required
for this process are shown. Efficient
initiation also requires the poly-A tail of the
mRNA bound by poly-A-binding proteins,
which in turn interact with eIF4G. In this
way, the translation apparatus ascertains
that both ends of the mRNA are intact
before initiating protein synthesis.

During the final phase of protein synthesis >, the binding of a

release factor to an A site bearing a stop codon (UAA, UAG, UGA)
terminates translation. They are not recognized by tRNA and do not
specify an amino acid. The release factor forces the peptidyl
transferase in the ribosome to catalyze the addition of a water
molecule instead of an amino acid, which frees the carboxyl end of
the growing peptide chain from its attachment to a tRNA molecule
the completed protein chain is released in to the cytoplasm. In a
series of reactions that requires additional proteins and GTP
hydrolysis, the ribosome then dissociates into its two separate
subunits.

Polyribosome: a series of ribosomes can

simultaneously translate the same
eukaryotic mRNA molecule.

Even though the genetic code is very universal, some variations can
take place. For example, mitochondrial DNA translates translate AUA
into methionine rather than isoleucine. A different type of variation,
called translation recoding, occurs in many cells: the other
nucleotide sequence information present in an mRNA can change
the meaning of the genetic code at a particular site in the mRNA
molecule. Bacteria, archaea and eukaryotes have a 21st amino acid
available that can be incorporated directly into a growing
polypeptide chain through translation recoding. Selenocysteine
contains a selenium atom in place of the sulfur atom of cysteine.
Selenocysteine is enzymatically produced from a serine attached to
a special tRNA molecule that base-pairs with the UGA codon (stop).
The mRNAs for proteins in which selenocysteine is to be inserted at

a UGA codon carry an additional nearby nucleotide sequence in the

mRNA that triggers this recoding event.
Inhibitors of prokaryotic protein synthesis are useful as antibiotics.
Many lodge in the pockets in the ribosomal RNAs and interfere with
the smooth operation of the ribosome others block specific parts
such as the exit channel.

Above, nonsense-mediated mRNA decay is shown. The failure to

correctly splice a pre-mRNA often introduces as premature stop
codon into the reading frame. These abnormal mRNAs are destroyed
by the nonsense-mediated decay mechanism. To activate this, an
mRNA molecule, bearing exon junction complexes (EJCs) to mark
successfully completed splices, is first met by a ribosome that
performs a test round of translation. As the mRNA passes through
the right channel of the ribosome, the EJCs are stripped off, and
successful mRNAs are released to undergo multiple rounds of
translation. However, if an in-frame stop codon is encountered
before the final EJC is reached, the mRNA undergoes nonsensemediated decay, triggered by the Upf proteins (green) that bind to
each EJC.

Translation of an mRNA sequence into an amino

acid sequence on the ribosome is not the end of
protein synthesis. To function, the polypeptide
chain must fold correctly into its 3D formation, bind
required cofactors, and assemble with its partner
protein chains. Noncovalent bond formation drives
these changes. Many proteins also require covalent
modifications of selected amino acids.

Cells utilize several types of chaperones. Many molecular

chaperones are called heat-shock proteins (hsp) because they are
synthesized in dramatically increased amounts after a brief
exposure of cells to an elevated temperature. This reflects the
operation of a feedback system that responds to an increase in
misfolded proteins (such as those produced by higher temperatures)
by boosting the synthesis of chaperones that help them refold. The
hsps share an affinity for the exposed hydrophobic patches of
incompletely folded proteins, and they hydrolyze ATP. The hsp70
machinery acts early in the life of many proteins, and the hsp60
proteins form a large barrel-shaped structure that acts after a
protein has been fully synthesized.

A cell makes several quality-control choices for a difficult-to-fold,

newly synthesized protein.

Proteasome: deliberately destroys aberrant proteins.

The proteasome cap recognizes proteins marked by a polyubiquitin

chain and subsequently translocates them into the proteasome
core, where they are digested. At an early stage, the ubiquitin is
cleaved from the substrate protein and is recycled. Translocation
into the core of the proteasome is mediated by a ring of ATPases
that unfold the substrate protein as it is threaded through the ring
and into the proteasome core.

The proteasome cap contains proteins that recognize and hydrolyze

ubiquitin and a hexameric ring through which ubiquitylated proteins
are threaded. Stable protein substrates may require hundreds of
cycles of ATP hydrolysis before they are successfully pulled through
the AAA protein ring.
Many proteins are controlled by regulated destruction. Activation of
a specific E3 molecule creates a new ubiquitin ligase. Eukaryotic
cells have many different
E3 molecules, each
activated by a different
signal. An exposed
degradation signal can be
created in the protein to be
degraded, which binds a
ubiquitin ligase, causing the
addition of a polyubiquitin
chain to a nearby lysine on
the target protein (see
below and beside).

< The
production of
a protein in a
eukaryotic
cell. The final
level of each
protein in a
eukaryotic
cell depends
upon the
efficiency of
each step
depicted.

If nucleic
acids are
required to
synthesize
proteins and
proteins are, in turn, required to synthesize nucleic acids, how did
such a system arise? One view is that an RNA world existed before
modern cells arose. RNA both stored genetic information and
catalyzed the chemical reactions in primitive cells. Only later did
DNA take over as the genetic material and did proteins become the
major catalysts and structural components of cells.

Comparisons of many RNA structures

have revealed conserved motifs, short
structural elements that are used over
and over again as parts of larger
structures.
Protein catalysts require a surface with
unique contours and chemical properties
on which a given set of substrates can
react. In the same way, an RNA molecule
with an appropriately folded shape can
serve as a catalyst ribozymes. They
work by positioning metal ions at their
active sites, giving them a wider range of
catalytic activities.

Synthetic ribozymes with properties to be studied are selected in

vitro. Beginning with a large pool of nucleic acid molecules
synthetizes in the laboratory, those rare RNA molecules that possess
as specific catalytic activity can be isolated and studied (shown
below). During the autophosphorylation step, the RNA molecules are
kept sufficiently dilute to prevent the cross-phosphorylation of
additional RNA molecules. Several repetitions of this procedure are
necessary to select the very rare RNA molecules.
Because RNA has all the properties required of a molecule that could
catalyze a variety of chemical reactions, including those that lead to
its own synthesis, it has been proposed that RNAs served long ago
as the catalysts for template-dependent RNA synthesis.

This hypothetical process would require

catalysis both of the production of a second
RNA strand of complementary nucleotide
sequence and the use of this second RNA
molecule as a template
to form many
molecules of RNA with
the original sequence.
The red rays represent
the active sites.