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DNA structure and synthesis
• DNA is a polymer of
nucleotides
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Nucleotide base terminology
Base
Nucleoside
Nucleotide
Adenine
Adenosine
Adenosine ___ Phosphate
Guanine
Guanosine
Guanosine____Phosphate
Cytosine
Thymine
Cytidine
Thymidine
Cytidine_____Phosphate
Thymidine____Phosphate
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Nucleotides generally have 1 (mono), 2 (di) or 3 (tri)
phosphate groups.
Nucleosides that make up DNA have deoxyribose instead of ribose
as sugar; have deoxy as a prefix in the name.
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• Which bases pair with each other?
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DNA
“backbones”:
alternating
sugarphosphates.
DNA chains are antiparallel.
http://courses.bio.psu.edu/fall2005/biol230weve
/tutorials/tutorial2_files/figure_16_5_part2.gif
Interior:
Complementary bases.
T always pairs
with A,
C with G.
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directionality
in DNA
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DNA chains held
together with H
bonds.
A-T pairs: 2
G-C pairs: 3.
Bases are flat, planar; they stack on the inside of the
molecule. Hydrophobic interactions stabilize DNA.
DNA chains twist together around
a central axis, not around each other.
http://genetics.nbii.gov/i
mages/BasePairs.gif
Structure/Function Relationships in DNA
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• Note how the structure presents a mechanism for
exact replication, needed for the genetic molecule.
• Bases can be arranged in any sequence; provides
info for specifying 20 amino acids.
• Mispairing due to mistakes, damage, lead to
mutation, lead to individual variation and evolution.
“It has not escaped our notice that
the specific pairing we have
postulated immediately suggests
a possible copying mechanism
for the genetic material."
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-Watson and Crick
http://www.sciencetechnologyaction.com/lessons2.php?s
tudyid=6&edition=1
DNA replication
• Origin of DNA replication: particular site on DNA where
copying of the DNA always starts.
– Replication is bidirectional
– In each direction, there is a replication fork.
– Most bacterial DNA is circular, so there is one Origin
and one terminus
• Replicon: a length of DNA molecule replicated after
initiation from one origin. Examples:
– Bacterial DNA, plasmids, segments of eukaryotic
chromosomes.
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E. coli, a typical impatient bacterium
• E. coli takes 30 minutes to replicate all its DNA, yet it can
double every 20 minutes. How does it do this?
• Starts a round of DNA replication before finishing the
previous round.
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A couple of words on terminology
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• A chemical reaction in which molecules are
combined to make a products is a synthesis reaction.
• DNA is synthesized in cells, but we can direct DNA
synthesis in a test tube also. PCR, sequencing both
involve DNA synthesis.
• DNA replication is a natural biological process in
which a DNA molecule is copied in a cell.
– Replication is a specific act of synthesis.
What every DNA polymerase needs
• A template of DNA
– Enzymes copy a single strand of DNA
– Can’t work without something to copy from
• A primer
– A primer is a polynucleotide with a “free 3´OH end”
– In normal DNA replication, this is RNA
• A substrate
– To make DNA, a polymer, monomers are needed
– Nucleotide triphosphates (NTPs) are the monomers
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Numbering of ring positions
Ring positions on nitrogenous
bases “use up” the numbers,
so positions on sugar are
indicated by “prime”.
5’ and 3’ positions on sugar are
very important.
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Adding and removing bases:
Directionality
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• DNA synthesis is ALWAYS in a 5´ to 3´ direction
– See next slide.
• All 3 DNA pols have a 3´ to 5´ exonuclease activity
– Nuclease: enzyme activity that cuts nucleic acids
– Exo- means cuts from an end
– 3´ to 5´ means the opposite direction from synthesis
• “proofreading” ability; polymerase can “backspace” to
remove a base put it by mistake.
• DNA pol I has a 5´ to 3´ exonuclease activity
– Cuts off DNA bases in same direction as synthesis
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**
*
*
Initiation of DNA replication
*
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* Helicases
unwind the
DNA
•DNA pol requires a primer to add to: Primase makes an RNA
•Synthesis is 5´ to 3´, and antiparallel.
•Leading strand; synthesis follows replication fork.
Problems due to antiparallel nature of DNA
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In this picture, replication of the
lower strand of DNA can
proceed as the “replication
fork” moves from right to left
because the direction of
synthesis of new DNA
is 5’ to 3’.
What about the other strand?
The one made without a hitch is
called the “leading strand”, the
other is the “lagging strand”.
Okazaki fragments
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Because of requirement for 5’to 3’ synthesis, lagging strand
must repeatedly top and start; needs an RNA primer each time.
Cleaning up Okazaki’s
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Ligase needed
DNA Pol I cuts out RNA primers, replaces them with DNA.
Uses both the 5´ to 3´ exonuclease and polymerase activities.
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About RNA
1) DNA is double stranded, but RNA is single
stranded.
However, RNA can base-pair with itself to
create double stranded regions.
RNA
DNA
tRNA
genetics.gsk.com/graphics/ dna-big.gif http://www.fhi-berlin.mpg.de/th/JG/RNA.jpg
http://www.santafe.edu/images/rna.gif
About RNA-2
2) RNA contains ribose instead of
deoxyribose
3) RNA contains uracil instead of
thymine.
www.layevangelism.com/.../ deoxyribose.htm
http://www.rothamsted.bbsrc.ac.uk/notebook/courses/guide/images/uracil.gif
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3 kinds of RNA
mRNA: a copy of the gene; is
translated to make protein.
tRNA: smallest RNA, does actual
decoding.
tRNA
rRNA: 3 sizes that, along
with proteins, make up a
ribosome.
rRNA
http://www.cu.lu/labext/rcms/cppe/traducti/tjpeg/trna.jpeg;
Tobin and Duschek, Asking About Life; http://www.tokyo-ed.ac.jp/genet/mutation/nort.gif
Transcription: making mRNA
• RNA a polymer assembled from monomers
– Ribonucleoside triphosphates: ATP, UTP, GTP,CTP
• RNA polymerase
– Multi-component enzyme
– Needs a template, but NOT a primer
– In bacteria, a component (sigma) recognizes the promoter
as the place on DNA to start synthesis
– Synthesis proceeds 5’ to 3’, just as in DNA
• mRNA is complementary and antiparallel to the DNA
strand being copied.
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Transcription-2
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• The order of nucleotides in the RNA reflects the
order in the DNA
• If RNA is complementary to one DNA strand, then
it is identical (except for T change to U) to the other
DNA strand.
Either DNA strand may
contain the gene!
Transcription just runs
the other direction.
Sense, antisense
Compare the sense strand of the DNA to the mRNA.
Note that mRNA synthesis will be 5’ to 3’ and antiparallel.
http://users.rcn.com/jkimball.ma.ultranet/BiologyPages/S/SenseStrand.gif
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Transcription needs a Promoter
A promoter is nontranscribed DNA
Prokaryotes
Eukaryotes
http://opbs.okstate.edu/~petracek/2002%20Gene%20expression/img043.gif
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The Process of Transcription
• Promoter recognition: 2 consensus sequences
– The -10 region: TATAAT (10 bases upstream from
where transcription actually starts.
– The -35 region, farther upstream, also important.
– “Consensus” sequence meaning the DNA sequence from
many genes averages out to this.
– The closer these 2 regions actually are to the consensus
sequences, the “stronger” the promoter, meaning the
more likely RNA polymerase binding and transcription
will occur.
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Consensus sequence
Numbers indicate the
percentage of different genes
in which that nucleotide
appears in that spot in the
promoter sequence.
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http://www.uark.edu/campus-resources/mivey/m4233/promoter.gif
The Process of Transcription-2
• After binding to the promoter, polymerase “melts”
DNA, lines up first base at the +1 site = Initiation.
• RNA synthesis continues (Elongation), only the
template strand being transcribed.
• Termination: must be a stop sign, right?
– In bacteria, hairpin loop followed by run of U’s in the
RNA. Of course, the DNA must code for complementary
bases and a run of A’s. See next.
– Termination factor “rho”. Accessory protein.
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Termination of Transcription in Bacteria
The hairpin loop
destabilizes the interactions
between the DNA, mRNA,
and polymerase; U-A
basepairs are very weak,
and the complex falls apart.
In euks, termination
occurs with a processing
step.
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http://www.blc.arizona.edu/marty/411/Modules/Weaver/Chap6/Fig.0649ac.gif
About mRNA structure, etc.
• Start site of transcription is NOT equal to start site
of Translation
– First codon read, AUG, is downstream from the first
ribonucleotides. +1 is transcription start, not translation
start.
– AUG marks the beginning of an Open Reading Frame
(ORF).
• Lifetime of a eukaryotic mRNA is variable
• For prokaryotes, mRNA is short lived, fits in with
need of microbes to respond quickly to changes in
environment.
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Storage and use of genetic information
• The genetic code
– Three bases (in a row) specify an amino acid
• Transcription
– The synthesis of a mRNA, complementary to one of the
DNA strands, containing the genetic code
• Translation
– Proteins and rRNAs in the ribosome along with tRNAs
translate the genetic code into proteins.
• Post-translational modification
– Proteins are altered after synthesis
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The Genetic Code
• Four bases taken how many at a time? Need to code
for 20 different amino acids.
– Each base = 1 amino acid: only 4
– Every 2 bases = 1 a.a.: 16 combinations, 4 short.
– Every 3 bases: 64 combinations, enough.
• Every 3 bases of RNA nucleotides: codon
– Each codon is complementary to 3 bases in one strand of
DNA
– Each codon (except for T →U switch) is the same as 3
bases in the other DNA strand.
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More about the Genetic Code
• The code is
– Unambiguous: each codon specifies 1 amino acid
– Degenerate: a particular amino acid can be coded for by
several different codons.
– Ordered: similar codons specify the same amino acid.
– Commaless, spaceless, and non-overlapping : each 3
bases is read one after the other.
– Punctuated: certain codons specify “start” and “stop”.
– Universal: by viruses, both prokaryotic domains, and
eukaryotes (except for some protozoa, mitochondria).
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The Genetic Code-2
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http://www.biology.arizona.edu/molecular_bio/problem_sets/nucleic_acids/graphics/gencode.gif
Wobble
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• Crick’s Wobble Hypothesis
• The code is “ordered”
– The first 2 positions are more important
– When lining up with the anticodon of the tRNA, the third
position doesn’t bind as tightly, thus a looser match is
possible.
– Because of this flexibility, a cell doesn’t need 61
different tRNAs (one for each codon).
• Bacteria have 30-40 different tRNAs
• Plants, animals have up to 50.
tRNA
3D structure:
The familiar loops
of the 2D structure
are labeled.
Decoder end:
Complementary to
codon.
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hto-b.usc.edu/~cbmp/2001/ tRNA/trna%20s1.jpg
3’ end:
Attaches to
amino acid.
Translation
•
•
•
•
mRNA: provides message to be translated.
Ribosomes: functional workbench for synthesis.
tRNA: bring aa to ribosome, decode mRNA.
Aminoacyl tRNA synthetases: enzymes that attach amino
acids to tRNAs.
• Protein factors: help move process along: initiation,
elongation, and termination.
• Process is similar, but different between prokaryotes and
eukaryotes.
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Initiation and Termination of protein synthesis
formyl
• AUG is always the first codon (initiator codon)
– Establishes an “open reading frame” (ORF)
– Ribosome begins synthesis with a methionine
• In bacteria, it is N-formylmethionine (fMet)
• After synthesis , either formyl group is removed or
entire fMet is removed (Met in eukaryotes)
• Three codons serve as termination codons:
– UGA, UAG, UAA; any one can be a stop signal
– Do NOT code for an amino acid
– Cause translation to end; protein is completed
Translation-1
• Initiation
– Small subunit, mRNA, met-tRNA, IFs, GTP
– mRNA: sequence for binding to ribosome needed
• prokaryotes: Shine-Delgarno
• Eukaryotes: Cap and Kozak sequence
– (GCC)RCCATGG where R is a purine
–
–
–
–
First tRNA is fMet-tRNA in prokaryotes
IFs are protein Initiation Factors
GTP needed for energy
When all have come together, Large subunit added
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Translation-2
• Ribosome has 3 sites
– AA site where tRNA-aa first sits in
– P site where tRNA with growing peptide sits
– E or Exit, site transiently occupied by used tRNA
• Elongation, with help of EFs and GTP
– tRNA with new aa sits in A site
– Stays in A site if anticodon on tRNA is complementary
to codon on mRNA.
– tRNA in P site transfers growing chain to new aa
• Catalyzed by rRNA
• Ribosome moves relative to mRNA and tRNAs
– 41tRNAs now in new sites, new codon lined up
Ribosome schematic
http://staff.jccc.net/pdecell/proteinsynthesis/translation/elongation12.gif
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Translation-3
• Termination
– When stop codon is in A site, no tRNA binds
– GTP-dependent release factor (protein) removes
polypeptide from tRNA in P site. All done.
– Ribosomal subunits typically dissociate.
• Do a Google Search for translation animation
– Many hits. Note presence, absence of E site
– Note shape of ribosomes
– Note whether role of rRNA in catalysis is shown
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Nonsense mutations and suppressors
• A mutation may change a
normal codon to a stop
codon; protein synthesis
ends prematurely.
(nonsense mutation)
A second mutation can cure the
original: a “suppressor”.
If the gene for a tRNA is
mutated in the anticodon so that
the stop codon is now read by
the tRNA.
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Polysomes and Polycistronic mRNA
• In eukaryotes, when mRNA enters the cytoplasm,
many ribosomes attach to begin translation. A
mRNA w/ many ribosomes attached = polysome.
• In eukaryotes, the mRNA for a single gene is
processed and translated; in prokaryotes, mRNA
can be polycistronic, meaning several genes are on
the same mRNA and are translated together
– With no nucleus, translation can begin in prokaryotes
before transcription is over.
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Polysomes
Multiple ribosomes attach to the mRNA and
begin translating.
Strings of ribosomes can be seen attached to
the mRNA.
http://opbs.okstate.edu/~petracek/Cha
pter%2027%20Figures/Fig%202729b-bottom.GIF
www.cu.lu/labext/rcms/
cppe/traducti/tpoly.html
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