Transcript Lectures 1 & 2 (2010.03.05 & 2010.03.06)

Chapter 4
DNA, RNA, and the flow of genetic
information
The Story of “Molecular Biology”
Watson and Crick (1953)
Structure of DNA & DNA Replication
DNA Structure
Molecular structure of nucleic acids; a structure for deoxyribose nucleic acid.
Watson, J.D., and Crick, F.H.
Nature (April 1953) 171(4356):737-738
DNA Replication
Genetical implications of the structure of deoxyribonucleic acid.
Watson, J.D., and Crick, F.H.
Nature (May 1953) 171(4361):964-967
Nobel Prize in Medicine (1962)
Evidence for the Structure of DNA
Evidence Available to Watson and Crick:
1. Nucleotides & Nucleotide Pairing (Pyrimidine + Purine)
2. Chargaff’s Rule (A = T, G = C)
3. X-ray Diffraction Analysis (Rosalind Franklin and Maurice Wilkins)
Rosalind Franklin
Molar Properties of Bases
Organism
Adenine
Thymine
Guanine
Cytosine
AT/GC Ratio
E. coli
26.0
23.9
24.9
25.2
1.00
D. pneumoniae
29.8
31.6
20.5
18.0
1.59
M. tuberculosis
15.1
14.6
34.9
35.4
0.42
Yeast
31.3
32.9
18.7
17.1
1.79
Sea Urchin
32.8
32.1
17.7
18.4
1.85
Herring
27.8
27.5
22.2
22.6
1.23
Rat
28.6
28.4
21.4
21.5
1.33
Human
30.9
29.4
19.9
19.8
1.52
Chargaff’s Rules:
1. T + C (pyrimidines) = A + G (purines)
2. T = A and C = G (because of “base-pairing”)
3. The amount of A + T is not necessarily equal to the amount of G + C
(“GC-rich” genomes)
Chargaff’s Rule
Another set of clues available to Watson and Crick came from the work done by
Erwin Chargaff, who studied the nucleotide composition of DNAs from different
organisms.
Chargaff established certain “rules” about the amounts of each component of DNA:
1. The total amount of pyrimidine nucleotides (T + C) always equals the total
amount of purine nucleotides (A + G).
2. The amount of T always equals the amount of A, and the amount of C always
equals the amount of G. But the amount of A + T is not necessarily equal to
the amount of G + C. This ratio varies among different organisms.
Wilkins and Franklin (1952)
The Antiparallel DNA Double Helix
The pairing of a purine with a pyrimidine
(A-T or G-C pair) is exactly the diameter
of the DNA double helix determined
from X-ray data.
DNA Replication
SEMI-CONSERVATIVE REPLICATION: 2 new DNA helices are produced
• Each helix has 2 DNA strands
• One strand is from the parental DNA (PURPLE)
• The other strand is newly synthesized (BLUE)
Nucleic Acids
Nucleic Acids
•
Nucleic acids store, transmit, and use genetic information.
•
DNA = deoxyribonucleic acid & RNA = ribonucleic acid
•
The monomers of nucleotides consist of a pentose sugar, a phosphate group,
and a nitrogen-containing base.
Nucleic Acids
• RNA has “ribose” & DNA has “deoxyribose”
• The “backbone” of DNA and RNA is a chain of sugars and phosphate groups,
bonded by PHOSPHODIESTER LINKAGE.
• The phosphate groups link carbon 3′ in one sugar to carbon 5′ in another sugar.
• The two strands of DNA run in opposite directions (antiparallel).
Deoxyribonucleosides
Ribonucleosides
Nucleotide Structure
•
Ring structures are found in both the base and the sugar
a. Base rings are numbered as usual
b. Sugar ring numbers are given the “prime” designation
•
Phosphodiester Bond: covalent bond between the sugar and phosphoryl group
•
-N-glycosidic Bond: covalent bond between the 1'-carbon of the sugar and a
nitrogen atom of the base
Phosphodiester Bond
Adenosine Triphosphate (ATP)
Adenosine Triphosphate
Adenosine Diphosphate
Adenosine Monophosphate
Adenosine
“Nucleoside” vs. “Nucleotide”
Nucleotide Nomenclature
DNA Double Helix
DNA Double Helix
•
BASE PAIRING: a non-covalent attraction aiding in maintaining the double helix
structure is hydrogen bonding between base pairs
a. Adenine forms 2 hydrogen bonds with Thymine: A = T
b. Cytosine forms 3 hydrogen bonds with Guanine: G ≡ C
•
The two DNA strands are COMPLEMENTARY strands - the sequence of bases on
one strand automatically determine the sequence of bases on the other strand
•
The chains run ANTIPARALLEL and this is the only structure that allows base
pairs to form the Hydrogen Bonds that hold them together
The DNA Double Helix
Single RNA Strand
Antiparallel DNA Strands
The Structure of DNA
DNA:
• 3’,5’-phosphodiester bridges link nucleotides
together to form polynucleotide chains.
• The 5’-ends of the chains are at the top; the
3’- ends are at the bottom.
The Structure of RNA
RNA:
• 3',5'-phosphodiester bridges link nucleotides
together to form polynucleotide chains.
• The 5'-ends of the chains are at the top; the
3'- ends are at the bottom.
The DNA Double Helix
A, B, Z form of DNA (table 4.2)
The Chromosome
The Eukaryotic Chromosome
Eukaryotes (number and size of chromosomes vary)
a. True nucleus enclosed by a nuclear membrane
b. Nucleosome which consists of a strand of DNA wrapped around a disk of
histone proteins (DNA appears like “beads-on-a-string”)
• String of beads then coils into a larger structure called the 30 nm fiber
• With additional proteins next coiled in to a 200 nm fiber
Beads-on-a-String
The Histones
Histone H4
Amino acid sequence of the amino-terminus of Histone H4.
Acetylation and deacetylation of lysine side chains. Histone acetylation
is linked to transcriptional activation and associated with euchromatin.
N-Acetyl
Lysine
The Histones
The Nucleosome
Each nucleosome is composed of a core particle plus
histone H1 and linker DNA. The nucleosome core particle
is composed of a histone octamer and about 146 bp of
DNA. Linker DNA consists of about 54 bp. Histone H1
binds to the core particle and to linker DNA.
Histone-Depleted Chromosome
DNA Replication
The Central Dogma of Molecular Biology
Replication
The two functions of DNA comprise the Central Dogma of Molecular Biology:
1. DNA can reproduce itself (replication)
2. DNA can copy its information into RNA (transcription)
3. RNA can specify a sequence of amino acids in a polypeptide (translation)
DNA Replication
DNA must be replicated before a cell divides, so that each daughter cell inherits a
copy of each gene
• Cell missing a critical gene will die
• Essential that the process of DNA replication produces an absolutely
accurate copy of the original genetic information
• Mistakes made in critical genes can result in lethal mutations
Structure of the DNA molecule suggests the mechanism for accurate replication
• An enzyme could “read” the nitrogenous bases on one strand of a DNA
molecule adding complementary bases to a newly synthesized strand
• SEMICONSERVATIVE Replication: one DNA strand is the original (PARENT)
strand and the other is newly synthesized (DAUGHTER) strand
• Compare to CONSERVATIVE and DISPERSIVE replication.
DNA Replication
SEMICONSERVATIVE REPLICATION: 2 new DNA helices are produced
• Each helix has 2 DNA strands
• One strand is from the parental DNA (PURPLE)
• The other strand is newly synthesized (BLUE)
Meselson-Stahl Experiment
14N
and 15N are stable (non-radioactive) isotopes,
but 14N is far more abundant in nature (99.6%).
Meselson-Stahl Experiment
• Centrifugation of DNA in a cesium chloride (CsCl)
gradient……a DENSITY gradient.
• Cultures grown for many generations in 15N and
14N media provide control positions for heavy
(15N) and light (14N) DNA bands.
• When the cells grown in 15N are transferred to a
14N medium, the first generation produces an
intermediate DNA band and the second
generation produces two bands: one
intermediate and one light.
Meselson-Stahl Experiment
What method of DNA replication?
The results of Meselsohn and Stahl’s
experiments prove that only the semiconservative model of DNA
replication is possible.
Semi-Conservative Replication:
1. A single intermediate (14N/15N
hybrid DNA) band is produced in
the first generation.
2. One intermediate (14N/15N hybrid
DNA) and one light (14N/14N DNA)
band in the second generation.
The Replisome
The Replication Machinery
1. Topoisomerase (phosphodiester bonds)
2. Single-Stranded DNA-Binding Protein
3. Helicase (hydrogen bonds)
4. RNA Primase
5. RNA Primer
6. Okazaki Fragment
7. DNA Polymerase III
8. DNA Polymerase I
9. DNA Ligase
Supercoiled DNA & Topoisomerases
The DNA molecule on the left is a relaxed closed circle; breaking the DNA helix and unwinding
it by two turns before re-forming the circle produces two SUPERCOILS.
Positively supercoiled (extra-twisted) regions accumulate ahead of the replication fork as the
parental strands separate for replication. This “supercoiling” can be created or relaxed by
TOPOISOMERASES.
Leading Strand vs. Lagging Strand
Events:
1. Helicase: “opening” of replication fork
2. ssDNA-binding protein
3. Primase: RNA Primer
4. Leading Strand : one RNA primer
5. Lagging Strand: Okazaki fragments
6. DNA Pol III: extension of primer
7. DNA Pol I: removes RNA primer
8. DNA Ligase: joins 3’ and 5’ ends
LEADING STRAND SYNTHESIS: a single RNA primer is produced at the replication origin.
DNA polymerase III catalyzes the addition of nucleotides in the 5'- 3' direction.
The Replication Fork
The two newly synthesized strands have opposite polarity. On the leading strand,
5'  3' synthesis moves in the same direction as the replication fork; on the
lagging strand, 5'  3' synthesis moves in the opposite direction.
Lagging Strand Synthesis
A short piece of RNA (BROWN) serves as a primer for the synthesis of each Okazaki
fragment. The length of the Okazaki fragment is determined by the distance
between successive RNA primers.
Lagging Strand Synthesis
Lagging Strand Synthesis
•
•
•
Many RNA primers are needed as the replication fork moves
DNA polymerase III catalyzes the elongation of the new strand (5'  3' direction)
Final steps:
a. The removal of the RNA primers by DNA Polymerase I
b. Filling in the gaps by DNA Polymerase I
c. Sealing the fragments into an intact strand of DNA by DNA Ligase
Summary of DNA Replication
•
The two DNA strands being replicated are ANTIPARALLEL to one another
a. DNA polymerase III can only catalyze in the 5'  3' direction
b. However, the replication fork moves in one direction with both strands
replicated simultaneously (LEADING & LAGGING Strands)
•
Small RNA primers are needed for a starting point of DNA replication
•
RESULT: there are different mechanisms for replication of the two strands
a. CONTINUOUS Replication: the leading strand is replicated in one long
segment
b. DISCONTINUOUS Replication: the lagging strand is replicated in many
small segments
Transcription
The Central Dogma of Molecular Biology
Replication
The two functions of DNA comprise the Central Dogma of Molecular Biology:
1. DNA can reproduce itself (replication)
2. DNA can copy its information into RNA (transcription)
3. RNA can specify a sequence of amino acids in a polypeptide (translation)
RNA Transcription in E. coli
Transcription of E. coli ribosomal RNA genes. The genes are being transcribed from
left to right. The nascent rRNA product associates with proteins and is processed by
nucleolytic cleavage before transcription is complete.
Classes of RNA Molecules
1. Messenger RNA (mRNA)
• mRNA directs the amino acid sequence of proteins
• A complimentary copy of a gene
• It has the codon for an amino acid in a protein
2. Ribosomal RNA (rRNA)
• Structural and functional component of the ribosome
• Forms ribosomes by reacting with proteins
• 3 types in prokaryotes
• 4 types in eukaryotes
3. Transfer RNA (tRNA)
• Transfers amino acids to the site of protein synthesis
The RNA Content of an E.coli Cell
Transcription
•
Transcription is catalyzed by RNA Polymerase
•
Produces a copy of only one DNA strand
•
Stages of transcription:
1. Initiation: RNA polymerase binds to the promoter region at the beginning
of the gene
2. Chain Elongation: formation of a 3‘  5' phosphodiester bond, generating
a complementary copy
3. Termination: the final step of transcription, the RNA polymerase releases
the newly formed RNA molecule
Stages of Transcription
Initiation
Elongation
Termination
Gene Orientation
The sequence of a hypothetical gene and the RNA transcribed from it are shown.
By convention, the gene is said to be transcribed from the 5' end to the 3' end, but
the template strand of DNA is copied from the 3' end to the 5' end. Growth of the
ribonucleotide chain proceeds 5‘  3'.
Post-transcriptional Processing of mRNA
•
Prokaryotes release a mature mRNA at the end of termination for translation
•
Eukaryote mRNA is a primary transcript which still must be processed in posttranscriptional modification, a three step process:
a. A 5' cap structure is added
• This structure is required for efficient translation of the final mRNA
b. A 3' poly(A) tail (100 to 200 units) is added by poly(A) polymerase
• Poly(A) tail protects the 3' end of the mRNA from enzymatic digestion
• Prolongs the life of the mRNA
c. Splicing
5'-Methylated Cap
Modification to the 5' end of the mRNA with 7-Methyl-Guanosine (m7G)
Polyadenylation
RNA Splicing
SPLICING: removal of portions of the mRNA transcript that are not protein coding
1. Bacterial chromosomes are “continuous” as all DNA sequences from the
chromosome is found in the mRNA
2. Eukaryotic chromosomes are “discontinuous”
• There are extra DNA sequences within the genes that do not encode
any amino acid sequence called INTRONS (intervening sequences)
• Presence of introns makes direct translation to synthesize proteins
impossible
3. The introns are cut out and the EXONS (coding sequences) are spliced
together
mRNA Splicing
mRNA Splicing & Post-Translational Modification
Triose phosphate isomerase gene from maize and the encoded enzyme. Diagram
of the gene showing 9 exons and 8 introns. Some exons contain both translated
and untranslated sequences.
Protein Translation
The Central Dogma of Molecular Biology
Replication
The two functions of DNA comprise the Central Dogma of Molecular Biology:
1. DNA can reproduce itself (replication)
2. DNA can copy its information into RNA (transcription)
3. RNA can specify a sequence of amino acids in a polypeptide (translation)
Classes of RNA Molecules
1. Messenger RNA (mRNA)
• mRNA directs the amino acid sequence of proteins
• A complimentary copy of a gene
• It has the codon for an amino acid in a protein
2. Ribosomal RNA (rRNA)
• Structural and functional component of the ribosome
• Forms ribosomes by reacting with proteins
• 3 types in prokaryotes
• 4 types in eukaryotes
3. Transfer RNA (tRNA)
• Transfers amino acids to the site of protein synthesis
Protein Synthesis
1. Protein synthesis is called TRANSLATION and is carried out on RIBOSOMES
• rRNA
• Proteins
2. Protein synthesis occurs in multiple places on one mRNA at a time
• mRNA plus the multiple ribosomes are called a POLYSOME
3. tRNA (with ANTICODON) recognizes the appropriate CODON on the mRNA
4. AMINOACYL tRNA SYNTHETASE
• An enzyme that catalyzes the “esterification” of a specific amino acid or its
to its partner tRNA to form an aminoacyl-tRNA
• Sometimes called “charging” the tRNA with the amino acid.
• Once the tRNA is charged, a ribosome can transfer the amino acid from the
tRNA onto a growing peptide, according to the genetic code.
The Genetic Code
The message on DNA that has been translated to mRNA:
1. Degenerate: more than one codon can code for the same amino acid
2. Specific: each codon specifies a particular amino acid
3. Non-Overlapping:
• None of the bases are shared between consecutive codons
• No non-coding bases appear in the base sequence
4. Universal: all organisms use the same code
The Genetic Code
•
•
•
Degenerate & Specific: 64 codons encode 61 amino acids & 3 “stop” signals
Multiple codes for an amino acid tend to have two bases in common
Codons are written in a 5'  3' sequence
Non-overlapping Code
a. In an overlapping code, each letter is part of three different three-letter.
b. In a non-overlapping code, each letter is part of only one three-letter word.
The Reading Frame
One mRNA contains three different reading frames. The same string of letters read
in three different reading frames will be translated into three different “messages”
or protein sequences. Thus, translation of the correct message requires selecting
the correct reading frame.
Schematic of the Translation Process
Ribosomes
Ribosome: the “workbench” of the cell; holds mRNA and charged tRNAs in the correct
positions to allow assembly of polypeptide chain.
Composed of rRNA + Proteins
• Two subunits (held together by ionic bonds and hydrophobic forces)
a. Small ribosomal subunit: 1 rRNA + 33 proteins
b. Large ribosomal subunit: 3 rRNA + 49 proteins
•
Polysome: many ribosomes on one mRNA when many copies of the protein
are being made simultaneously
Ribosome Structure
Large subunit has three tRNA binding sites:
1. A (aminoacyl) site binds with anticodon of charged tRNA
2. P (polypeptide) site is where tRNA adds its amino acid to the growing chain
3. E (exit) site is where tRNA sits before being released from the ribosome.
Ribosome Structure
Eukaryotic vs. Prokaryotic Ribosomes
The prokaryotic and eukaryotic ribosomes consist of two subunits, each of which
contains ribosomal RNA and proteins. The large subunit of the prokaryotic
ribosome contains two molecules of rRNA: 5S and 23S. The large subunit of
almost all eukaryotic ribosomes contains three molecules of rRNA: 5S, 5.8S, and
28S. The sequence of the eukaryotic 5.8S rRNA is similar to the sequence of the 5'
end of the prokaryotic 23S rRNA.
Ribosome Structure
Sites for tRNA binding in prokaryotic ribosomes. During protein synthesis, the P
(“polypeptide”) site is occupied by the tRNA molecule attached to the growing
polypeptide chain, and the A site holds an “aminoacyl-tRNA”. The growing
polypeptide chain passes through the tunnel of the large subunit.
tRNA
•
There is at least one tRNA for each amino acid to be incorporated into a protein
•
tRNA is single-stranded molecule with about 80 nucleotides
•
The overall structure is called a “cloverleaf”
a. Intrachain hydrogen bonding (A=U and G=C) occurs to give:
• Regions called stems with an -helix
• A type of L-shaped tertiary structure
b.
The 3'-OH group of the terminal nucleotide can covalently bind the
amino acid
c.
3 nucleotides at the base of the cloverleaf serve as the “anticodon”,
which forms hydrogen bonds to a “codon” on mRNA
tRNA
(A) The cloverleaf structure, a convention used to show the complementary base-pairing (red lines).
The ANTICODON is the sequence of three nucleotides that base-pairs with a CODON in mRNA. The
amino acid matching the codon/anticodon pair is attached at the 3′ end of the tRNA. tRNAs contain
some unusual bases, which are produced by chemical modification after the tRNA has been
synthesized. For example, the bases denoted Ψ (pseudouridine) and D (dihydrouridine) are derived
from uracil. (B and C) Views of the actual L-shaped molecule, based on X-ray diffraction analysis. All
tRNAs have very similar structures. (D) The linear nucleotide sequence of the molecule, color-coded
to match A, B, and C.
Tertiary Structure of tRNA
The cloverleaf-shaped molecule folds into this three dimensional shape. The tertiary
structure of tRNA results from base pairing between the TC loop and the D loop, and
two stacking interactions that (left) align the TC arm with the acceptor arm, and
(right) align the D arm with the anticodon arm.
The Genetic Code
The CODONS are always written with the 5′-terminal nucleotide to the left. Note
that most amino acids are represented by more than one codon, and that there are
some regularities in the set of codons that specifies each amino acid. Codons for
the same amino acid tend to contain the same nucleotides at the first and second
positions, and vary at the third position. UAA, UAG and UGA codons do not specify
any amino acid but act as termination sites (STOP CODONS), signaling the end of
the protein-coding sequence. AUG acts both as a START CODON, signaling the start
of a protein-coding message, and also as the codon that specifies METHIONINE.
Base Pairing at the Wobble Position
Base Pairing at the Wobble Position
The tRNAAla molecule with the anticodon “IGC” can bind to any one of three
codons specifying alanine (GCU, GCC, or GCA) because I can pair with U, C, or A.
Note that the RNA strand containing the codon and the strand containing the
anticodon are antiparallel.
Transcription and Translation
Protein Translation
1. Initiation
• Initiation factors (proteins), mRNA, initiator tRNA, and small and large
ribosomes come together
• Ribosome has two sites to bind tRNA
a. P site binds to the growing peptide
b. A site binds the aminoacyl tRNA
2. Chain Elongation: a three step process
•
An aminoacyl tRNA binds to A site
•
Peptide bond formation occurs catalyzed by peptidyl transferase
•
Translocation (movement) of ribosome down the mRNA chain next to
codon
a. Shifts the new peptidyl tRNA from the A site to the P site
b. Chain elongation requires hydrolysis of GTP to GDP
Protein Translation
3. Termination
• Upon finding a “stop” codon a release factor binds the empty A site
• The bond between the last amino acid and peptidyl tRNA is hydrolyzed
releasing the protein
• The protein released may not be in its final form
• Post-translational modification may occur before a protein is fully
functional
a. Cleavage
b. Association with other proteins
c. Bonding to carbohydrate or lipid groups
The Polysome (Polyribosome)
Several ribosomes can work together to translate the same mRNA, producing
multiple copies of the polypeptide. A strand of mRNA with associated ribosomes is
called a POLYSOME (or Polyribosome).
Inhibitors of Protein Synthesis
Two important purposes to biochemists:
•
Inhibitors have helped solve the mechanism of protein synthesis
•
Those that affect PROKARYOTIC but not EUKARYOTIC protein synthesis are
effective ANTIBIOTICS
•
Streptomycin: an aminoglycoside antibiotic that induces mRNA misreading;
the resulting mutant proteins slow the rate of bacterial growth
•
Puromycin: binds at the A site of both prokaryotic and eukaryotic ribosomes,
accepting the peptide chain from the P site, and terminating protein synthesis
Inhibitors of Protein or RNA Synthesis
INHIBITOR
EFFECT
Acting only on Bacteria (Anti-bacterial)
Tetracycline
Streptomycin
Chloramphenicol
Erythromycin
Rifamycin
blocks binding of aminoacyl-tRNA to A-site of ribosome
prevents the transition from initiation complex to chain-elongating
ribosome and also causes miscoding
blocks the peptidyl transferase reaction on ribosomes (Step 2)
blocks the translocation reaction on ribosomes (Step 3)
blocks initiation of RNA chains by binding to RNA polymerase
(prevents RNA synthesis)
Acting on Bacteria and Eukaryotes
Puromycin
Actinomycin D
causes the premature release of nascent polypeptide chains by its
addition to growing chain end
binds to DNA and blocks the movement of RNA polymerase
(prevents RNA synthesis)
Acting only on Eukaryotes
Cycloheximide
blocks the translocation reaction on ribosomes (Step 3)
Anisomycin
blocks the peptidyl transferase reaction on ribosomes (Step 2)
α-Amanitin
blocks mRNA synthesis by binding preferentially to RNA polymerase II
Puromycin is a Protein Synthesis Inhibitor
Formation of a peptide bond between puromycin at the A site of a ribosome and
the nascent peptide bound to the tRNA in the P site. The product of this reaction is
bound only weakly in the A site and dissociates from the ribosome, thus
terminating protein synthesis and producing an incomplete, inactive peptide.