09 18 2006 MCB Basic Molecular Genetic Mechanism
09 18 2006 MCB Basic Molecular Genetic Mechanism
09 18 2006 MCB Basic Molecular Genetic Mechanism
• Transcription
• RNA Processing
• mRNA Translation
• DNA Replication
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.
Glycosidic
bond
1
Table 4-1. Naming Nucleosides and Nucleotides
Phospho
-diester
bond
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:
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Nobel Laureate
Physiology or Medicine 1962:
DNA Structure
B-DNA
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.
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Sequence-specific recognition
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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
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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.
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RNA Tertiary Structures
2000
1974 1996
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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)
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.
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Structural Basis of Transcription: An RNA Polymerase II Elongation Complex at
3.3 Angstrom Resolution (Science 2001, 292, 1876-1882)
Nucleic acids in the transcribing complex and their interactions with pol II.
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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.
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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.
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.
34 Angstrom
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The helix-turn-helix DNA binding motif in lambda Cro bound to DNA
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Control of Gene Expression in Eukaryotes
100~ 200 bp
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
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TBP binds in the minor groove and induces large structural
changes in DNA
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.
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.
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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.)
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.
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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.
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During Chain Elongation Each Incoming
Aminoacyl-tRNA Moves through Three
Ribosomal Sites
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.
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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)
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
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