Chapter 4: Basic Molecular Genetic Mechanisms

Four basic molecular

genetic processes:

• Transcription
• RNA Processing
• mRNA Translation
• DNA Replication

Instructor: 袁小琀 Tel: (02) 27884151

Hanna S. Yuan E-mail: hanna@sinica.edu.tw

References: 1. “Molecular Cell Biology” 5th Edition, W. H. Freeman and Company, USA, Chapter 4.
2. “Introduction to Protein Structure” 2nd Edition, by Branden, C. and Tooze, J. G,
Garland Publishing, Inc. USA, Chapter 6, 7.

Structure of Nucleic Acids The chemical structures of the principal

bases in nucleic acids.
The primary structures of both DNA
and RNA are linear polymers composed
of monomers called nucleotides.
All nucleotides have a common structure.

Glycosidic
bond

RNA: sugar is ribose

DNA: deoxyribose

1
 Table 4-1. Naming Nucleosides and Nucleotides

Nucleoside: a base and a sugar

Nucleotide: nucleoside with one, two or three phosphate groups.
Nucleoside monophosphate: contains a single phosphate
Nucleoside diphosphate: contains a pyrophosphate group
Nucleoside triphosphate: contains a third phosphate. Used in synthesis of nucleic acids.
ATP also serves as energy carrier.
GTP plays a role in intracellular signaling and acts as an energy reservoir.

A nucleic acid strand is a linear polymer composed of nucleotides with end-to-

end directionality.
Native DNA is a double helix of complementary antiparallel strands.

Phospho
-diester
bond

Watson-Crick base pairs

Stick diagram of the chemical structure of double-
helical DNA, unraveled to show the sugar-phosphate
backbones (sugar rings in green), base-paired bases
(light blue and light red), and hydrogen bonds
between the bases (dark red dotted lines).
Space-filling model of B-DNA. the most common
form of DNA in cells. The sugar and phosphate
residues (gray) in each strand form the backbone,
which is traced by a red line, showing the helical
twist of the overall molecule.

2
 X-Ray Fiber diffraction

Based on the works of X-ray fiber diffraction on the low humidity A-form
DNA, Watson and Crick suggested the first model of double-stranded
DNA in 1953.
Diffraction patterns of
DNA, A-DNA (left half)
and B-DNA (right half):
The simulated diffraction pattern of helices in fiber
crystallites:

3
Nobel Laureate
Physiology or Medicine 1962:

CRICK, FRANCIS HARRY COMPTON

Great Britain, Institute of Molecular Biology

WATSON, JAMES DEWEY

U.S.A., Harvard University

WILKINS, MAURICE HUGH FREDERICK

Great Britain, University of London

"for their discoveries concerning the

molecular structure of nuclear acids
and its significance for information
transfer in living material”

DNA Structure

B-DNA

A-DNA Base pairs are

displaced off axis-
major grooves are
deeper and minor
grooves are shallower.
Z-DNA
Left-handed.
CGCGCG-
first structure
solved in 1979.

B-DNA: Base pairs stack on the axis and perpendicular to the helix axis-
grooves are of equal depth. The first single X-ray structure of
CGCGAATTCGCG was solved in 1980 in Richard Dickerson’s lab.

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A-DNA occurs as a dehydrated form of DNA, the A-form is also the preferred conformation
of RNA. Z-DNA can occur at stretches of alternating G and C bases and may be important
for controlling replication. But by far the most important physiological conformation is B-
DNA.

Since the base pairs are attached asymmetrically to the backbone, one groove between the
strands is wider than the other. These are called the major and the minor groove. Both
grooves provide opportunities for base-specific interactions, but the major groove is better
suited for that task and more often observed as the primary binding site for proteins.

Major groove and minor groove

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Sequence-specific recognition

W: recognition sites at major groove

S: recognition sites at minor groove

Specific and non-specific interactions between proteins and DNA

This is well illustrated, when

one considers the hydrogen
bond donors, hydrogen bond
acceptors and methyl groups,
accessible at the bottoms of
the major and minor grooves
of B-DNA. The functional
groups provide ample
opportunity for sequence-
specific interaction of
proteins with DNA.
Additionally, the idealized B-
DNA backbone is quite
significantly distorted in real-
life and the phosphate
D A A A D A backbone conformation itself
A A D will thus display sequence
A D A
information.

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 Proteins prefer to bind DNA at major grooves likely because:
(1) more space and
(2) more chemical features
presented by the major groove.

Ideal B-DNA
Rise = 3.4 Angstrom

Roll > 0o bends towards major groove

Roll < 0o bends towards minor groove

Twist < 34o, DNA is unwound

Twist > 34o, DNA is over wound

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The denaturation and renaturation of double-stranded DNA molecules.

(a) If both strands are closed circles, denaturation disrupts the double helix, but the two single strands
become tangled about each other and cannot separate. (b) If one or both strands are nicked, however,
the two strands will separate on thermal denaturation.

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 Light absorption and temperature in DNA denaturation. (a) Melting of
doubled-stranded DNA can be monitored by the absorption of ultraviolet light at
260 nm. As regions of double-stranded DNA unpair, the absorption of light by
those regions increases almost twofold. The temperature at which half the bases in
a double-stranded DNA sample have denatured is denoted Tm (for temperature of
melting). Light absorption by single-stranded DNA changes much less as the
temperature is increased. (b) The Tm is a function of the G·C content of the DNA;
the higher the G·C percentage, the greater the Tm.

RNA Secondary and Tertiary Structures

RNA has a hydroxyl group at the 2’

position, making it more chemical
labile than DNA. RNA can be easily
hydrolyzed in alkaline solution.

Most cellular RNAs are single-

stranded and exhibit a variety of
conformation.

Some RNA, called ribozymes, have

catalytic activity.

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 RNA Tertiary Structures
2000
1974 1996

Science, 2005, 309, 1508-1514.

Transcription- A template DNA strand is transcribed into a complementary

RNA chain by RNA polymerase

Transcription of DNA into RNA is catalyzed by +1: the site at which

RNA polymerase, which can initiate the RNA polymerase
synthesis of strands de novo on DNA starts transcription
templates.
1. The ribonucleotide to be added at the 3’ end
of a growing RNA strand is specified by base
pairing between the next base in the template
DNA strand and the incoming
ribonucleotiside triphosphate (rNTP).
2. The nucleotide at the 5’ end of an RNA
strand retains all three of its phosphate groups;
3. all subsequent nucleotides release
pyrophosphate (PPi) when added to the chain
and retain only their a phosphate (red). The
released PPi is subsequently hydrolyzed by
pyrophosphatase to Pi, driving the equilibrium
of the overall reaction toward chain
elongation.
4. In most cases, only one DNA strand is
transcribed into RNA.

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Stages in Transcription

Transcription movie

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Structural model of the bacterial
RNA polymerase bound to a
promoter (Open complex)

Bacterial RNA polymerase

contains five subunits: α, α,
β, β’, ω.

Template DNA strand is

shown in grey and
nontemplate strand is
shown in purple. A Mg2+
ion at the active site is
shown as a gray sphere.

DNA is bent sharply after

its entry into the enzyme.

Yeast RNA Polymerase Structure

Yeast RNA Polymerase II at 2.8 Å Resolution

The clamp is in an open state, allowing entry of straight promoter DNA for the initiation
of transcription. Three loops extending from the clamp may play roles in RNA unwinding
and DNA rewinding during transcription. Two metal ions at the active site, one
persistently bound and the other possibly exchangeable during RNA synthesis.

Ref: Science, 2001, 292, 1863-1876.

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 Structural Basis of Transcription: An RNA Polymerase II Elongation Complex at
3.3 Angstrom Resolution (Science 2001, 292, 1876-1882)

Comparison of structures of free

pol II (top) and the pol II transcribing
complex (bottom). The clamp
(yellow) closes on DNA and RNA,
which are bound in the cleft above
the active center. The remainder of
the protein is in gray.
Structure of the pol II transcribing complex. The
template DNA strand is in blue, nontemplate strand
in green, and RNA in orange. The Rpb1 bridge
helix traversing the cleft is highlighted in green.
The active site metal A is shown as a pink sphere.

Nucleic acids in the transcribing complex and their interactions with pol II.

The main technical challenge of this work was the isolation

and crystallization of a transcribing complex. Initiation at an
RNA polymerase II promoter requires a complex set of
general transcription factors and is poorly efficient in
reconstituted systems. Moreover, most preparations contain
many inactive polymerases, and the transcribing complexes
obtained would have to be purified by mild methods to
preserve their integrity. The initiation problem was overcome
with the use of a DNA duplex bearing a single-stranded “tail”
at one 3’-end. Pol II starts transcription in the tail, two to three
nucleotides from the junction with duplex DNA, with no
requirement for general transcription factors. All active
polymerase molecules are converted to transcribing complexes,
which pause at a specific site when one of the four nucleoside
triphosphates is withheld. The problem of contamination by
inactive polymerases was solved by passage through a heparin
column; inactive molecules were adsorbed, whereas
transcribing complexes flowed through, presumably because
heparin binds in the positively charged cleft of the enzyme,
which is occupied by DNA and RNA in transcribing
complexes.
(A) DNA (tailed template) and RNA sequences. DNA template and non-template strands are in blue and green,
respectively, and RNA is in red.
(B) Ordering of nucleic acids in the transcribing complex structure. Nucleotides in the solid box are well ordered.
Nucleotides in the dashed box are partially ordered, whereas those outside the boxes are disordered. Three protein
regions that abut the downstream DNA are indicated.

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• Duplex DNA is seen entering the main cleft of the enzyme and unwinding before the active site. Nine
base pairs of DNA-RNA hybrid extend from the active center at nearly right angles to the entering
DNA, with the 3’ end of the RNA in the nucleotide addition site.
• The 3’ end is positioned above a pore, through which nucleotides may enter and through which RNA
may be extruded during back-tracking.
• The 5’-most residue of the RNA is close to the point of entry to an exit groove. Changes in protein
structure between the transcribing complex and free enzyme include closure of a clamp over the DNA
and RNA and ordering of a series of ‘switches’ at the base of the clamp to create a binding site
complementary to the DNA-RNA hybrid.
• Protein- nucleic acid contacts help explain DNA and RNA strand separation, the specificity of RNA
synthesis, ‘abortive cycling’ during transcription initiation, and RNA and DNA translocation during
transcription elongation.

Organization of Genes in DNA Differs in Prokaryotes and Eukaryotes

• In prokaryotic DNA, related protein-coding genes are clustered into a functional
region, an operon, which is transcribed from a single start site into one mRNA encoding
multiple proteins. Translation of a mRNA can begin before synthesis of the mRNA is
complete.
• In eukaryotic DNA, each protein-coding gene is transcribed from its own start site,
and very often the coding regions (exons) are separated by noncoding regions (introns).
The primary RNA transcript produced from such a gene must undergo processing to
yield a functional mRNA.

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Eukaryotic Primary RNA Transcripts Are Processed to Form Functional mRNAs

RNA Processing
The primary RNA transcript produced
from such a gene must undergo
processing to yield a functional
mRNA.
1. During processing, the ends of all
primary transcripts are modified by
addition of a 5’ cap and 3’ poly(A)
tail;
2. Many transcripts also undergo
splicing, removal of the introns and
joining of the exons.

Structure of the 5’ methylated cap of eukaryotic

mRNA. The distinguishing chemical features are the 5’ → 5’
linkage of 7-methylguanylate to the initial nucleotide of the
mRNA molecule and the methyl group on the 2’ hydroxyl of the
ribose of the first nucleotide (base 1). Both these features occur in
all animal cells and in cells of higher plants; yeasts lack the
methyl group on base 1. The ribose of the second nucleotide (base
2) also is methylated in vertebrates.

Overview of RNA processing in eukaryotes using β-globin gene as an example

The β-globin gene contains three protein-coding exons (red) and two intervening noncoding
introns (blue).
• Transcription of this and many other genes starts slightly upstream of the 5’ exon and
extends downstream of the 3’ exon, resulting in noncoding regions (gray) at the ends of
the primary transcript. These regions, referred to as untranslated regions (UTRs), are
retained during processing.
• The 5’ 7-methylguanylate cap (m7Gppp; green dot) is added during formation of the
primary RNA transcript, which extends beyond the poly(A) site.
• After cleavage at the poly(A) site and addition of multiple A residues to the 3’ end,
splicing removes the introns and joins the exons.

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Control of Gene Expression in Prokaryotes
Regulation of Transcription from lac
operon of E. coli. Lac operon encodes
three enzymes required for the
metabolism of lactose.

Transcription activators bind next to

promoter and increase the rate of
transcription initiation by RNA
polymerase.

Repressors bind to the operator

sequence which overlap or lie adjacent
to promoters. Binding of a repressor
to an operator inhibits transcription
initiation.

The DNA-binding activity of most

Movie- lac operon regulationanimations\a04-03-
lac_regulation.swf
bacterial repressors is modulated by
small effector molecules (inducer).
This allows bacterial cells to regulate
transcription of specific genes in
response to change in the
concentration of various nutrients in
environment.

Lambda Cro Lambda repressor

34 Angstrom

The first structure of a DNA-

binding protein, Lambda Cro,
was reported by Brian
Matthews’s lab in 1981.

1. Both are dimers

2. α2 and α3 are the helix-turn-helix motifs
3. α3 helices are separated by a distance of 34 Angstrom

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The helix-turn-helix DNA binding motif in lambda Cro bound to DNA

The first structure of a

protein/DNA complex, 434
repressor/DNA, was solved by
Steve Harrison’s lab in 1987.

Small molecules regulate expression of many bacterial genes via DNA

binding repressor by allosteric control
Allosteric effectors bind at sites distant from functional binding sites but
cause a conformational change which alters the DNA binding affinity.

Trp repressor regulates the synthesis of L-Trp by a “negative

feedback loop”. When L-tryptophan binds to Trp repressor,
the heads change positions to produce the active form of the
repressor, which binds to DNA.

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 Control of Gene Expression in Eukaryotes

100~ 200 bp

~ 1000 to >20,000 bp away

Transcriptional
Initiation Complex

Movie- examples of
transcription initiation

Steven Burley
reported the
crystal structure of
TATA-box binding
protein in 1992

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 Crystal structure
of the TATA-box
binding protein
with a saddle-like
topology

20 Angstrom

In 1993, the crystal

structure of TATA-box
binding protein in
complex with DNA was
resolved in Paul Sigler
(yeast) and Steven
Burley’s (Arabidopsis)
laboratories.

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TBP binds in the minor groove and induces large structural
changes in DNA

TATA box region

DNA is bended for ~ 100 degree

Phenylalanine intercalates
between base pairs
producing sharp kinks

Positive roll:
Helix bends toward
major groove

Low twist:
Helix unwound
Protein-DNA interactions:
-Mainly hydrophobic
-H-bonds only at center. The extra –NH2 at
the minor groove of GC base pairs might
interfere with binding.
-TATA sequence is more flexible for bending.

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 Translation The Three Roles of RNA in Protein Synthesis

Messenger RNA (mRNA)- carries the genetic information copied from DNA in the form of a
series of three-base code "words," each of which specifies a particular amino acid.

Transfer RNA (tRNA) is the key to deciphering the code words in mRNA. Each type of amino
acid has its own type of tRNA, which binds it and carries it to the growing end of a polypeptide
chain if the next code word on mRNA calls for it.

Ribosomal RNA (rRNA) associates with a set of proteins to form ribosomes. These complex
structures, which physically move along an mRNA molecule, catalyze the assembly of amino
acids into protein chains.

Messenger RNA Carries Information from DNA in a Three-Letter Genetic Code

Of the 64 possible codons in the genetic code, 61 specify individual amino acids and three are stop
codons.
Synthesis of all protein chains in prokaryotic and eukaryotic cells begins with the amino acid
methionine. In most mRNAs, the start (initiator) codon specifying this aminoterminal methionine
is AUG.

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The Folded Structure of tRNA Promotes Its Decoding Functions

The partially unfolded molecule is commonly depicted as a cloverleaf. All tRNAs have a
similar three-dimensional structure that includes an acceptor arm for attachment of a
specific amino acid and a stem-loop with a three-base anticodon sequence at its ends. The
anticodon can base-pair with its corresponding codon or codons in mRNA.

Nonstandard Base Pairing Often Occurs between Codons and Anticodons

The first and second bases in an

30-40 tRNAs in bacterial cells and 50-100 in animal cells.
mRNA codon form Watson-Crick
base pairs with the third and second
bases, respectively, of a tRNA
anticodon. However, the base in the
third (or wobble) position of an mRNA
codon often forms a nonstandard base
pair with the base in the first (or
wobble) position of a tRNA anticodon.
Wobble pairing allows a tRNA to
recognize more than one mRNA codon
(top); conversely, it allows a codon to
be recognized by more than one kind
of tRNA (bottom), although each
tRNA will bear the same amino acid.
Note that a tRNA with I (inosine) in
the wobble position can "read"
(become paired with) three different
codons, and a tRNA with G or U in the
wobble position can read two codons.
Although A is theoretically possible in
the wobble position of the anticodon, it
is almost never found in nature.

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 Aminoacyl-tRNA Synthetases Activate Amino Acids by Linking Them to tRNAs

Each of the 20 aminoacyl-tRNA synthetases recognizes a single amino acid and covalently
links it to a cognate tRNA, forming an aminoacyl-tRNA. This reaction activates the amino
acid, so it can participate in peptide-bond formation.

The two-step aminoacylation reaction requires energy from the hydrolysis of ATP. The equilibrium of
the overall reaction favors the indicated products because the pyrophosphate (PPi) released in step 1 is
converted to inorganic phosphate (Pi) by a pyrophosphatase. The 3’ end of all tRNAs, to which the
amino acid attaches, has the sequence CCA. Class I synthetases (purple) attach the amino acid to the 2
hydroxyl of the terminal adenylate in tRNA; class II synthetases (green) attach the amino acid to the 3
hydroxyl. (Ad = adenine; Cyt = cytosine.)

Ribosomes Are Protein-Synthesizing Machines

The general structure of ribosomes in prokaryotes and eukaryotes. In all cells, each ribosome consists
of a large and a small subunit. The two subunits contain rRNAs of different lengths, as well as a different
set of proteins. All ribosomes contain two major rRNA molecules (dark red)- 23S and 16S rRNA in
bacteria, 28S and 18S rRNA in eukaryotes- and one or two small RNAs (light red). The large subunit of
vertebrate ribosomes also contains a 5.8S rRNA based-paried to the 28S rRNA.
S: svedlberg unit, a measure of the sedimentation rate.

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 The AUG Start Codon Is Recognized by Methionyl-tRNAiMet

Binds at P site

Binds at A site

•Two types of methionine tRNA are found in all cells. One, designated
tRNAiMet, is used exclusively to start protein chains, and the other, designated
tRNAMet, delivers methionine to internal sites in a growing protein chain. In
bacteria, a formyl group (CHO) is added to methionyl-tRNAiMet, forming fMet-
tRNAiMet.

•The same aminoacyl-tRNA synthetase (MetRS) charges both tRNAs with

methionine. But only Met-tRNAiMet (i.e., activated methionine attached to
tRNAiMet) can bind at the appropriate site on the small ribosomal subunit (the P
site) to begin synthesis of a protein chain. (The regular tRNAMet binds only to
another ribosomal site, the A site.)

Bacterial Initiation of Protein

Synthesis Begins Near a Shine-
Dalgarno Sequence in mRNA
Bacterial initiation of protein synthesis.
The three initiation factors (IF1, IF2, and IF3)
bind to the small subunit of the ribosome. The
30S preinitiation complex, together with the
fMet-tRNAiMet, binds the mRNA, guided by the
Shine-Dalgarno sequence, which is
complementary to the 3’ terminus of the 16S
rRNA. The AUG sequence is then located, from
which protein initiation will begin. Addition of
the large (50S) ribosomal subunit is
accompanied by release of the protein factors
and hydrolysis of IF2-GTP to yield the complete
70S initiation complex. The charged initiator
tRNA (fMet-tRNAiMet) is positioned in the P site.
The ribosome now is ready for entry of the next
aminoacyl-tRNA at the A site, which will lead
to the formation of the first peptide bond. The
initiation process is similar in archaeans, except
there is no formylation of the Met-tRNAiMet.

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Eukaryotic initiation of protein synthesis

Establishment of the initiation complex must follow a precise sequence of events, and
the large and small ribosomal subunits must be kept apart until late in the process. Two
eukaryotic factors, eIF3 (a large multimeric protein with about eight subunits) and eIF6
fulfill this role.

Initiation of translation in eukaryotes

1. A 40S preinitiation complex is formed when a

ternary complex of eIF2 bound to GTP and
Met-tRNAiMet associates with the small (40S)
ribosomal subunit, which is complexed with two
other factors, eIF3 and eIF1A, that stabilize
binding of the ternary complex.
2. The 5' cap (m7G) of the mRNA to be translated
is guided to the preinitiation complex by a
subunit of the multiprotein eIF4 complex, which
also unwinds any secondary structure at the 5'
end of the mRNA.
3. Scanning of the mRNA to position the initiator
tRNA at the AUG start codon.
4. Association of 60S large subunit. eIF5, assists
union of the 40S complex with the 60S subunit.
Factors eIF5 and eIF2 are GTP-binding
proteins, whose bound GTP is hydrolyzed
during translation initiation. The final 80S
initiation complex has a Met-tRNAiMet bound
at the P site.

25
During Chain Elongation Each Incoming
Aminoacyl-tRNA Moves through Three
Ribosomal Sites

Once the 80S ribosome with Met-tRNAi Met in the

ribosome P site is assembled:

1. The second aminoacyl-tRNA with EF1α-GTP is

bound to the A site.

2. GTP hydrolysis induces a conformational change if

the incoming aminoacyl-tRNA correctly base-pairs
with the second codon of the mRNA.

3. A peptide bond is formed between the incoming

amino acid and the growing chain at the P site. This
peptidyltransferase reaction is catalyzed by the large
rRNA.

4. Ribosome translocated along the mRNA a distance

equal to one codon. Hydrolysis of GTP in EF2-GTP
causes another conformational change in the mRNA Movie
and shifts the unacylated tRNAiMet to the E site .

Protein Synthesis Is Terminated by Release Factors When a Stop Codon Is

Reached

Termination of translation. When a ribosome bearing a nascent protein chain reaches a stop
codon (UAA, UGA, UAG), release factors (RFs) enter the ribosomal complex, probably at or
near the A site. In bacteria, RF1 or RF2 first recognizes a stop codon. RF3-GTP then catalyzes
cleavage of the peptide chain from the tRNA and release of the two ribosomal subunits,
reactions that require hydrolysis of GTP.

26
High resolution crystal structures of ribosomes are resolved

Movie: translation

The structure of the 70S ribosome shown was facilitated by atomic structures of the 30S and 50S subunits of
Thermus thermophilus.
(A) Two views of the 70S ribosome complexed with mRNA and tRNA, with the “top” view on the left and the
view from the 30S side on the right.
(B) Exploded view of the 50S (left) and 30S (right) subunits in the 70S ribosome, showing the locations of A-,
P-, and E-site tRNAs. (REF: Cell, 2002, 108, 557-572)

Simultaneous Translation by Multiple Ribosomes and Their Rapid Recycling

Increase the Efficiency of Protein Synthesis

The poly(A)-binding
protein I (PABI) and the
two components of yeast
eIF4 (4G and 4E) can
interact on mRNA to
circularize the molecule.

Model of protein synthesis on circular polysomes and recycling of ribosomal subunits. Multiple
individual ribosomes can simultaneously translate a eukaryotic mRNA, shown here in circular form
stabilized by interactions between proteins bound at the 3’ and 5’ ends. When a ribosome completes
translation and dissociates from the 3’ end, the separated subunits can readily find the nearby 5’ cap
(m7G) and initiate another round of synthesis. This rapid recycling of subunits on the same mRNA
contributes to the formation of polysomes and increases the efficiency of translation.
Movie- Transcription- translation

27