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Central dogma II -- Transcription
• DNA -> RNA -> protein
• But for retroviruses, DNA can also be
made by reverse transcription of RNA.
• To understand the lifecycle of a
retrovirus, we need to know more about
how DNA is replicated and transcribed, and
how RNA is translated into protein.
Vocabulary
• Replication -- copying DNA before cell division
• Transcription -- making an RNA copy
(messenger RNA or mRNA) of DNA.
Note -- Transcription involves copying in the same language
(e.g., court transcription).
• Translation -- making a protein from the
mRNA.
Note -- The nucleic acid language is being translated into the protein
language.
DNA ---------> DNA
transcription
translation
DNA ---------> RNA ----------> Protein
reverse
RNA --------->
DNA
transcription
replication
Transcription: DNA --> RNA
• DNA replication ensures that genetic
information is passed on unchanged from a
cell to its descendents.
• The major thing cells do with genetic
information is use it to encode PROTEINS.
• Every cell contains all of an organism’s
genes, so each cell could (in theory) make
every protein. But which proteins are made
is determined by which genes are copied
into RNA.
Cells express appropriate levels of proteins by regulating
transcription and translation
Two amplification steps: one gene can
make many RNA copies, and one RNA can
be translated into many proteins.
RNA is a nucleic acid similar to DNA, but with critical functional differences
The sugar in RNA contains an additional
OH group, so is ribose, not deoxyribose.
RNA uses uracil instead of thymine.
Uracil can still basepair with Adenine.
Most important:
RNA can basepair
with RNA or with
single-stranded
DNA, but it
is normally
single-stranded
rather than
double-stranded.
Single stranded RNA can fold into complicated
3D shapes resulting from intramolecular basepairing
07_05_RNA.jpg
Hairpin structures result from regions
of sequence that are complementary
to each other (inverted repeats).
Structure of a
ribozyme, an RNA
enzyme
Clicker question
When complementary RNA bases in an inverted
repeat pair with each other, they form which
type of structure?
1)
2)
3)
4)
5)
6)
Parallel double helix
Anti-parallel double helix
Parallel b-sheet
Anti-parallel b-sheet
-helix
Any of the above are possible
Clicker question
When complementary RNA bases in an inverted
repeat pair with each other, they form which
type of structure?
1)
2)
3)
4)
5)
6)
Parallel double helix
Anti-parallel double helix
Parallel b-sheet
Anti-parallel b-sheet
-helix
Any of the above are possible
Clicker question
3D structures of RNA are easier to predict than
3D structures of proteins.
1)
2)
True
False
Base-pairing makes prediction of RNA
structure reasonably accurate
• Many web-available structure prediction programs
e,g., http://www.santafe.edu/~pth/rna.html
• Can design and make DNA-based origami using single
stranded DNA: http://www.dna.caltech.edu/~pwkr/
Giant DNA complexes, imaged with an atomic force microscope (measures surface
topography). False color used to indicate height. Scale: 100s of nanometers.
Images taken from Paul Rothemund’s website: http://www.dna.caltech.edu/~pwkr/
The predictability of Watson-Crick base pairing
allows design of DNA-based digital logic circuits
• Erik Winfree’s lab at Caltech has designed and
demonstrated function of AND, OR, and NOT gates,
signal restoration, amplification, feedback and
cascading.
• We may use DNA circuits someday instead of, or in
addition to, silicon-based electronics.
Seelig et al., 2006, “Enzyme-free Nucleic Acid Logic Circuits, Science 314: 1585-1588.
See webpage for the Molecular Programming Project
www.ist.caltech.edu/mpp/
DNA is transcribed into RNA by
the enzyme RNA polymerase
Note that RNA
polymerase unwinds a
short region of the
DNA double helix.
Clicker question
This electron micrograph shows many molecules of RNA
polymerase simultaneously transcribing two adjacent genes.
The RNA polymerase molecules are moving
1) Right to left
2) Left to right
3) Not enough information to determine
“Nearly every major process in a
cell is carried out by assemblies of 10 or
more protein molecules… each of these
protein assemblies interacts with
several other large complexes… Indeed,
the entire cell can be viewed as a
factory that contains an elaborate
network of interlocking assembly lines,
each of which is composed of a set of
large protein machines.”
Bruce Alberts, Cell 92:291 (1998)
Eukaryotic gene transcription -Activation begins at a distance
Cramer, P. et al. (2000)
Science 288:640
Transcription video from DNA interactive
Clicker question
It normally takes a large amount of free energy to separate DNA
strands -- e.g., you would need to heat DNA to nearly boiling
temperature in the lab to separate strands. RNA polymerase
works at 37˚C, so how is it able to unwind the DNA in a closed
circular bacterial chromosome while it transcribes?
1)
2)
3)
4)
RNA polymerase has an associated helicase, which uses the energy of
ATP to force the DNA to unwind.
The RNA-DNA helix has a lower free energy than a DNA helix, so the
formation of the RNA-DNA helix displaces the other DNA strand.
RNA polymerase degrades one of the DNA strands, so that the
remaining strand can pair with the RNA strand.
Bacterial DNA is negatively supercoiled (i.e., underwound so that it has
more than 10.4 basepairs/turn), therefore unwinding a short stretch of
double helical DNA is energetically favorable.
Double stranded DNA can wrap around
itself to form supercoiled structures
To convert between 1 & 2 requires
breaking and rejoining of a bond.
This can be accomplished by a
topoisomerase enzyme.
No breaking of bonds is required to
convert between 2 & 3.
2
1
3
Change in the
linking number
No change in the
linking number
The topology of DNA is studied by mathematicians.
http://en.wikipedia.org/wiki/DNA_supercoil
RNA polymerase II movie
Roger Kornberg received the 2006 Nobel Prize in Chemistry
for studies on the “molecular basis of eukaryotic transcription”.
Comparison of RNA and DNA polymerases
• Both catalyze similar chemical reactions: formation of
phosphodiester bonds linking nucleotides.
– RNA polymerase links ribonucleotides.
Final product is single-stranded RNA.
– DNA polymerase links deoxyribonucleotides.
Final product is double-stranded DNA.
• RNA polymerase error rate: 1/104 nucleotides
DNA polymerase error rate: 1/107 nucleotides
Which is potentially most serious,
errors made by
1)
2)
3)
4)
5)
6)
DNA polymerase
RNA polymerase
Ribosome
Bi1 students
Bi1 TAs
Bi1 professors
Three major types of RNA
• mRNA (messenger RNA) codes for
proteins.
• rRNA (ribosomal RNA) forms part of the
ribosome, a machine involved in translation
of RNA into protein. (Next lecture)
• tRNA (transfer RNA) binds amino acids
and bring them to the ribosome during
protein translation.
(Next lecture)
Signals in DNA tell RNA polymerase
where to start and stop transcription
• Synthesis starts at a promoter,
a conserved sequence 5’ of a gene.
• Chain elongation occurs until
RNA polymerase encounters a
termination sequence on the DNA.
• RNA polymerase releases the
single stranded RNA and the
double stranded DNA template
upon encountering a termination
sequence.
• This slide depicts a bacterial
RNA polymerase. Transcription
initiation in eukaryotic cells is
more complicated.
Promoter and terminator sequences in
bacteria
Clicker question
Propose a mechanism by which the highlighted areas
could cause transcription to stop.
1)
2)
3)
4)
5)
The highlighted sequences contain stop codons.
RNA polymerase stops at pink sequences.
The highlighted sequences contain DNA mismatches, which
destabilize RNA polymerase.
The first highlighted sequence contains a binding site for a
termination factor, which knocks RNA polymerase off the DNA.
The first two sequences are inverted repeats of each other,
thus a hairpin, which is a signal for termination.
Clicker question
Propose a mechanism by which the highlighted areas
could cause transcription to stop.
5)
The first two sequences are inverted repeats of each other, thus a hairpin,
which is a signal for termination.