Chapter 12 DNA Structure, Replication, and Organization
DNA as the hereditary molecule;
• 1928: Frederick Griffith; infected mice with S and R strain streptococcus and found that were able to
obtain genetic information from other bacteria by transformation.
o Determined that carbohydrates not carrier of genetic information.
• 1942, Avery, Macleod and McCarthy; decided to find genetic material by growing bacterial culture and
used different enzymes to break down each component (carb, RNA, DNA, Protein).
o Determined that since when breakdown DNA= no transformation, DNA is carrier of genetic
• 1952, Hershey and Chase; used bacteriophages with radioactive labeling, one batch with radioactive
protein, and one batch with radioactive DNA.
o Determine DNA is transferred, since radioactivity was passed through DNA.
• DNA consists of four different nucleotides that consist of a fivecarbon
deoxyribose sugar, a triphosphate group and 1 of 4 nitrogenous bases.
o Purines (double rings): adenine and guanine.
o Pyrimidine (single carbon ring): thymine, cytosine.
o Chargaff’s Rule: # of purines = # pyrimidines; A=T and G=C
o Complemetary base pairing: A=T, G=C.
• DNA are made of dNTP = deoxynucleoside triphosphate i.e. dATP, dCTP,
o Nucleotide = Nucleoside + phosphate.
deoxyadenosine, deoxyguanosine, deoxythymidine,
• Nucleotides are linked to form polynucleotides with a sugar phosphate
backbone, where each phosphate is the link b/w the 3’ carbon of one sugar
and the 5’ carbon of the next one; Phosphodiester bond.
• Antiparallel, meaning that at one end is the phosphate bound to 5’ carbon
and the other end is a hydroxyl group bonded to 3’ carbon.
• 1953, Watson, Crick and Franklin: DNA has a doublestranded helical
structure in a righthanded way, 2 nm in diameter, separation of 0.34 to 3.4 nm in repeated intervals.
Eukaryotic DNA Organization;
• Two major types of chromosomal protein are associated with DNA structure and regulation; 70% of
chromatin are chromosomal proteins (37.5% histones and 37.5% nonhistones), 15% DNA, 10% RNA:
Small +ve charged that are attracted to –ve phosphates of DNA.
Five types of histones: H1, H2A, H2B, H3 and H4.
To pack DNA molecule into nucleus, can pack 2m of DNA into 10μm.
Nucleosome consists of nucleosome core and linker.
Nucleosome core: consists of 2 molecules each (8 total) of H2A, H2B, H3 and H4 which
combine to form a beadlike (electron microscope), for 2 turns of DNA.
• 147 base pairs of DNA wrapped around octamer of histone molecules.
Linkers link each nucleosome.
Diameter of nucleosome is 10nm; called 10nm chromatin fibre.
H1 histone binds to both nucleosome and linker DNA to compact DNA more, causes
coiled structure called 30 nm chromatin fibre or solenoid.
• Solenoid is inactive DNA; approx.. 6 nucleosomes per turn.
In interphase nuclei, chromatin fibers differentiate into Euchromatin (loosely
packed) and Heterochromatin (dense, different). o Barr body example of heterochromatin.
In mitotic/meiotic cell, fold and pack into thick, rodlike, visible chromosomes.
Regulate gene activity.
Proteins associated with DNA, other than histones.
Alter accessibility to gene by loosening histone binding (acetylases) or tighten histone
Others alter gene regulation i.e. transcription factors bind to promoter region to enhance
or repress a gene.
Prokaryotic Gene Regulation:
• Most have a single circular bacterial chromosome, but others have two or more and some are linear.
• Do not have nucleosome, but have charge proteins that form nucleoid.
o Suspended in plasma, no “nuclear membrane”.
• Many contain other DNA called plasmids that replicate independently of chromosome.
• SemiConservative replication: The two chains unwind & separate, each “old” strand is a template for
the addition of complementary bases, The result is two DNA helices that are exact copies of the parental
DNA molecule with one “old” strand & one “new” strand.
o 1958; Meselson and Stahl: used heavy N in different bacterial cultures then placed in light N 14
medium and allowed to divide for generations; DNA is extracted and centrifuged and viewed
ratios of heavy, light and medium DNA.
• DNA Polymerases are the primary enzymes of DNA replication.
o Deoxynucleoside triphosphates are substrate for DNA polymerases.
o As nucleotide approaches new strand, the beta and gamma phosphates
(pyrophosphate) are removed which allows phosphodiester linkage.
o Adds nucleotide to 3’ end of parent strand (3’ OH is always exposed, 5’
phosphate at old end).
o Events of DNA replication:
1. The two strands unwind
2. DNA polymerase can only add nucleotides to existing chain
3. Overall direction of new synthesis is in the 5’ to 3 direction, which
is antiparallel to that of the template strand
4. Nucleotides enter into a newly synthesized chain according to the A
T and GC complementary pairing rules
• Major Enzymes of DNA Replication:
o Helicase: unwinds DNA helix.
o SingleStranded Binding Proteins: stabilize single stranded DNA and prevent two strands at
the replication fork from reforming doublestranded DNA.
o Topoisomerase: avoids twisting of the DNA ahead of the replication fork by cutting DNA.
*in bacterial chromosome (circular)
o RNA Primase: assembles RNA primers in the 5’3’ direction to initiate new DNA strand.
o DNA Polymerase III: main replication enzyme in E. coli; extends the RNA primer by adding
DNA nucleotides to it.
o DNA Polymerase I: uses 5’3’ exonuclease to remove RNA of previously synthesized Okazaki
fragment, and uses 5’3’ polymerization to replace the removed RNA nucleotides with DNA
o Sliding Clamp: tethers DNA polymerase to the DNA template.
o DNA Ligase: seals nicks between adjacent bases after RNA primers
replaced with DNA. • Since synthesizes in 5’3’ direction, one strand must be leading (continuous) and the other must be
• Primase initiates by adding primer, and then DNA polymerase III adds DNA nucleotides, then
polymerase I removes and replaces nucleotides in lagging strand. Ligase then covalently bonds
nucleotides at the nick
• 500 to 1000 nucleotide/sec in prokaryotes, 50100 per sec in eukaryote
at the ori
Ys form a
• Replication origin is activated only once during the S phase, so no DNA is replicated twice
• Telomerase prevents shortening chromosome shortening in linear chromosome:
o At 3’ end of chromosome primer is removed, and since at end cannot be replaced
o Most eukaryotic genes have buffers, telomeres, which are hundreds to thousands of repeated
sequence, so will be unaffected until runs out.
o Telomerase adds DNA to ends of chromosome (kind of polymerase)
o Usually not active in somatic cells, so only capable of certain number of mitotic divisions
o Telomerase is active in embryo, and gametes since rapid divisions
o Explain how cancer cells can continuously replicate w/o being limited to certain number of
divisions. When cells develop into cancer cells their telomerase are reactivated, preserving
chromosome length. ▯if can turn off then cancer cells would eventually degrade so may cure
Prokaryotic DNA Replication:
• Replication from a single origin of replication
• Rolling circle model of replication: Two cells conjugate, One of double strands of plasmid (F factor)
breaks and moves into recipient, then replicated continuously in donor and discontinuously in recipient
Accuracy of DNA Replication: 6
• Polymerase is very accurate, if makes mistake it is a basepair mismatch (1 in 10 )
o Before adding another nucleotide it backs up, removes mispairing and then continues
• DNA repair mechanisms
o If mismatched pair, then distorted 2nm width is detected, and repair enzymes scan and will
remove sections, then polymerase adds correct nucleotides, then ligase seals
Chapter 1 Gene structure and Expression
Transcription (DNA RNA) : Information encoded in DNA is made into complementary RNA copy
• In prokaryotic cells, RNA polymerase synthesizes an mRNA molecule that is immediately available for
translation on ribosomes
• In eukaryotes, RNA polymerase synthesizes a precursormRNA containing extra segments that are
removed into translatable mRNA; exits nucleus through pore and is translated in cytoplasm
• Genetic code is written in three letter words using a four letter alphabet
o G, C, A, T r3presents DNA nucleotides; G, C, A, U represent RNA nucleotides
o Must be 4 , which can code for the 20 amino acids
o A triplet of nucleotides is called a codon o DNA; mRNA; polypeptide
• Genetic Code:
o Written in 5’3’ direction as they appear in mRNAs
o AUG= methionine, which is the start/initiator codon
o UAA, UAG, UGA are stop codons (nonsense/termination codon)
o Only methionine and tryptophan are specified by a single codon; rest of amino acids can be
represented by two or more codons this is called degeneracy/redundancy
o Commaless; sequential with no indicators between codons; only one correct reading frame for
o Universal; same codons in all living thins, means present very early in history
• Only one of the two DNA nucleotide strands acts as a template for synthesis of a complementary copy,
instead of both as in replication
• Only small region of the strand which codes for a certain gene serves as the template
• RNA polymerases catalyze the assembly of nucleotides into a RNA strand
• RNA molecules that arise are single stranded
• Gene consists of two main parts: promoter which is a control
sequence, and a transcription unit which is the section that is
copied into a RNA molecule
• Occurs in three steps:
1. Initiation: machinery assembles at promoter and begins
synthesizing RNA copy of gene
2. Elongation: RNA polymerase moves along the string of
nucleotides in a sequence of single stranded DNA
template & changes this information into a string of
nucleotides of single stranded premRNA
3. Termination: transcription ends, RNA molecule and
polymerase are released
• Similarities and Differences in eukaryotes and prokaryotes:
o Gene organization is the same, specific promoter
sequence is different
o In eukaryotes, RNA polymerase II cannot bind directly to
DNA; only once transcription factor binds to DNA
o In prokaryotes there are two forms of terminators which trigger termination
• RNA polymerase II: proteincoding genes, RNA polymerase III: tRNA and one rRNA, RNA polymerase
I: three other rRNAs
• Note, once an RNA polymerase has started transcription & has moved out of the way of the promoter,
another molecule of RNA polymerase may start creating another preRNA
Processing of mRNAs in Eukaryotes
• mRNAs contain noncoding regions that play key roles in protein synthesis
o In Prokaryotes, at 5’ untranslated region (5’ UTR), and 3’ UTR
o In Eukaryotes, gene is transcribed into precursor mRNA (premRNA)
o Soon after RNA polymerase II transcribes gene, a 5’ Guanine cap is added to premRNA
Cap protects the mRNA from degradation and is recognition site for ribosomes
o No terminator sequence on DNA, so near the 3’ end is a polyadenylation signal and cleave the
transcript just past this point
o Poly(A) polymerase adds 50250 adenine nucleotides; poly(A) tail
Protects from RNAdigesting enzymes in cytoplasm
o Introns interrupt protein gene sequence, they are removed from premRNA and not present in
mature mRNA; whatever is retained in mature mRNA is an exon o mRNA splicing removes introns from premRNAs and joins exons together
Occurs in a spliceosome, a complex of premRNA and small ribonucleoprotein
particles (complex of protein and RNA); known as snRNPs (snurps)
• Consist of small nuclear RNA (snRNA)
The splicesome cleaves
the intron at its end &
splices together the two
The cleaved intron &
Splicesome snRNPs are released.
1) cleaves exon/intron boundary
2) forms lariat
3) cleaves intron/exon boundary
• Introns provide selective advantage to organisms by increasing coding capacity by alternative splicing;
o In certain tissue, or environmental conditions, different regions of a premRNA may be identified
as introns and removed in different combinations to produce different mature mRNAs
o Regions that are exon in one situation may be removed as an intron in another situation
o αtropomyosin premRNA can be spliced to form smooth muscle or skeletal muscle
All introns are removed in both pathways, but exons 3, 10, and 11 are removed to
produce smooth muscle mRNA, and exons 2 and 12 are also removes to produce skeletal
• Human genome project reported that the human genome (nuclear & mtDNA) codes for~ 25,000 genes;
due to alternative splicing # of proteins able to far exceed this number of genes
Translation (mRNA Protein): assembly of amino acids into polypeptides on ribosomes
• In prokaryotic organisms occurs throughout cell, in eukaryote in cytoplasm
• mRNA: 100s of nucleotides long, template for translation, read in a 5’→ 3’ orientation
• tRNA: 7590 nucleotides long, internal anticodon sequence is complementary to mRNA codon, attached
to 3’ end is an amino acid specific to the anticodon, distinctive structure
o can base pair with themselves to wind into four doublehelical segments, forming a cloverleaf in
o Folds in 3dimensions in Lshape
o Anticodon is a threenucleotide segment that basepairs with a codon in mRNA
o Other end of the clover is a free 3’ that links to the amino acid corresponding to anticodon
Anticodon and codon are antiparallel; codons written 5’3’, anticodon 3’5’
o Wobble Hypothesis: complete set of 61 sense codons can be read by fewer than 61 distinct
tRNAs because of flexibility of allowing different 3 nucleotide of codon
o Adding an amino acid to tRNA is called aminoacylation/charging; creates an aminoacyl
tRNA, which is synthesized by aminoacyltRNA synthetases
Since has free energy (charging), will drive formation of peptide bond
• Ribosomes are rRNAprotein complexes that are protein assembly machines
o Ribosomes are ribonucleoprotein particles that translate RNA into amino acid chains
o Made up of large and small ribosomal subunits, composed of rRNA and ribosomal proteins
• Three Steps: o Initiation: Assembly of all the translation components on the start codon of the mRNA
The methioninetRNA with GTP binds to small ribosomal subunit
The above complex binds to the 5’ cap of the mRNA, and moves along the mRNA
(scanning) looking for AUG start codon in the P site
• This provides correct reading frame, called open reading frame
Large ribosomal subunit binds and GTP is hydrolyzed, now ready for elongation
o Elongation: Reading the string of codons in the single stranded mRNA & changing this
information into a polypeptide
Psite can only bind to peptidyltRNA
An aminoacyltRNA binds to Asite;
peptidyl transferase cleaves the amino
acid from the tRNA in the Psite and
forms a peptide bond between it and the
amino acid on the tRNA in the A site;
ribosome translocates the mRNA
moving the empty tRNA into the E site
and the peptidyltRNA into the Psite;
once next amino acid is bonded the
empty tRNA in the E site is released
and this continues until reaches
GTP→GDP + Pi provides the energy
when an aninoacyltRNA binds to A site
Binding of tRNA to appropriate site I facilitated by an elongation factor that is released
after binding is complete
13 cycles per second in eukaryotes, 1520 cycles/sec in prokaryotes
o Termination: Releasing the string of AA from the ribosome. Disassembly of the ribosome
subunits & mRNA
UAA, UAG, or UGA arrives in A site; a release factor (RF or termination factor)
which is a protein cannot basepair so the ribosome dissembles
• Once a ribosome has translated enough of the mRNA, there is space for another ribosome to begin
translating the same mRNA
o Polysomes consist of a series of ribosomes translating the same strand of mRNA
o Range from 1 or 2 for short mRNA, to 100s for longer mRNA
• Since there is no nuclear envelope in prokaryotes, as soon as mRNA is transcribed it is likely already
covered with ribosomes translating it
• Most proteins are inactive once released from polypeptide:
o Most proteins initially made are not functional as they require posttranslational processing;
folding, cleavage, modifications, binding to other proteins
o Chaperonins fold proteins into correct conformation
o Initial polypeptide may be processed into different mature proteins; another way of increasing
number of proteins encoded by few genes
• All proteins originate on ribosome in cytosol, but three final destinations:
2. Endomembrane system: ER, Golgi, lysosomes, secretory vesicles, nuclear envelope, plasma memb.
3. Other membrane bound organelles: nucleus,
• Proteins that are needed in the endomembrane system
have a signal sequence signal peptide) o Cotranslational import is the simultaneous import and translation of peptide
o Signal emerges from ribosome, then signal recognition particle binds and translation stops, then
SRP binds to SRP receptor, translation resumes, polypeptide enters lumen and binds to peptidase,
peptidase cleaves signal peptide from signal polypeptide and is released in lumen.
• Proteins needed in nucleus, mitochondria, chloroplasts etc. are sorted by posttranslational import;
Have short transit sequences; if going to nucleus have nuclear localization signals
Chapter 14 Control of Gene Expression
• Human egg when released from the ovary is almost completely metabolically inactive
• Within seconds of the egg & the sperm meeting, rapid cell division
• Mitosis produce cells of the body
• Cells differentiate into specialized cells with different functions
• Every nucleated cell of the body contains the same DNA template and genes
• Structural & functional differences in cell types result from the presence or absence of the products
resulting from expressed genes rather than the actual presence of genes themselves on DNA
• Gene must be expressed in the correct tissue at the correct time; very complex system
• Gene expression (transcription &/or translation) is like music played by an orchestra
o Gene is present in DNA but is it “On” or “Off”
o Gene is individually fine tuned
o Gene’s tuning is dynamic & aware of it surrounding
o If done properly you get music (normal development)
o If not, you get noise (abnormal/lethal development)
Regulation of Gene Expression in Prokaryotes
• Simple, single celled organisms with generation times in minutes; Rapid & reversible alterations so they
can adapt quickly to changes in their environment
• Genes are organized into a functional unit called an operon; cluster of prokaryotic genes and DNA
sequences involved in their regulation
o Promoter is the region where RNA polymerase begins transcription
o Operator is a short segment that is a binding sequence fir a regulatory protein
o Some operons are controlled by regulatory protein called a repressor; this reduces likelihood
that gene will be transcribed
o Other operons are controlled by activator which increases transcription of gene
o The cluster of genes in an operon is called a transcription unit
• The Lac operon is composed of promoter, operator, lacZ (Bgalactosidase), lacY (permease) and lacA
o When there is no lactose present a repressor is able to bind to the operator and therefore
polymerase cannot bind to the promoter
o When lactose is present it is converted to allolactose which can bind to the repressor, which
changes its shape so it can no longer bond to the operon; allactose is a inducer
o The lac operon is also controlled by positive regulatory systems; sensitive to availability of
glucose through the binding of CAP which binds upstream of promoter
o CAP bends DNA so more accessible to polymerase
o CAP only binds to DNA when binded with cAMP, and cAMP levels are inversely proportional to
the amount of glucose available
• The trp operon turned off in presence of tryptophan, the tryptophan acts as a corepressor by binding to
the repressor and blocking polymerase
o If tryptophan is not present, then no corepressor is available and the DNA will be translated by
polymerase and the 5 genes will be copied
Regulation of Gene Expression in Eukaryotes • More complicated since nuclear DNA is bound to histones thus need chromatin remodeling to loosen
histone DNA interaction (acetylases add acetyl groups to histones) or slide nucleosomes away from the
gene’s promoter region
• Shortterm regulation regulates genes which are quickly turned on or off in response to environment
(similar to prokaryotic); longterm regulation involves regulatory events required for an organism to
develop and differentiate
• Gene expression in eukaryotes is regulated at the transcriptional level (most occurs), posttranscriptional,
translational and posttranslational levels
• DNA that does not encode mRNA: Introns, Promoters, enhancers, Intergenic sequence, Repeats,
• Organization of eukaryotic proteincoding gene: Transcription unit is the segment that is transcribed into
premRNA, it contains 5’ UTR, exons, introns and 3’ UTR. Upstream is the promoter which contains a
o Transcription factors
recognize and bind to
TATA box and then
recruit polymerase II;
o Immediately upstream
is the proximal region,
promoter proximal elements which may bind to regulatory proteins that can stimulate or inhibit
o Further upstream is the enhancer; this contains regulatory sequences that may also bind to