!

"#
!"#$%&" (
)"*+,#-$,./0 $&-/1#&,*$,./0 $&-/1+-$,./
CHEM 590C, FaII 2011
Introduction to ChemicaI BioIogy
!"!
2%3-&4.$,# 567 ,1 *-#3-8"9 ,/ #:&.;.1.;"1
The human genome has 3 biIIion base pairs of DNA
Two meters of DNA must be stored in a micrometer-sized ceII
Need mechanism for compaction that stiII aIIows access
CompIex of DNA and protein is caIIed _________
"___________"
~200 bp of DNA
associated with octamer of 4 histone proteins:
(two moIecuIes each of H2A, H2B, H3 and H4)
!"$
567 *-#3-8,/8
Histones - rich in __________, which
interact with phosphodiesters of DNA
TeIomere
TeIomere
Centromere
!"%
<.;;./ #:&.;.1.;" "+";"/$1
RepIication origin
Prokaryotes - _____ repIication origin
Eukaryotes - Iarge (thousands of bp) and difficuIt to
identify; _______ origins on each chromosome
Centromere
Site at which the two chromatids
touch; anchor point for spindIe
formation during ceII division
TeIomeres
Ends of eukaryotic chromosomes
Some papers on the propriety of "prokaryotes:
Pace, N. R., Nature 2006, 441, 289 ()
Whitman, W. B., J. Bacteriol. 2009, 191, 2000 (+)
Pace, N. R. J. Bacteriol. 2009, 191, 2006 ()
Pace, N. R. J. Bacteriol. 2009, 191, 2008 ()
Nature 2006, 441, 289
!"&
=;-8"1 .> /%#+".1.;"1
from Molecular Biology of the Cell
!"'
<:&.;.1.;"1
Each human ceII (with a few exceptions) contains two
copies of each chromosome, one inherited from the
father and one inherited from the mother.
FemaIes have two X chromosomes, and maIes
have one each X and Y chromosomes.
(22 x 2) + 2 = __ chromosomes
Numbered roughIy
according to size
!"(
?"/.;" $. *&.$",/
OnIy 1-2% of eukaryotic DNA codes for proteins!
Large stretches of non-coding regions within each gene:
Average gene size is 27,000 bp
1,300 bp required to encode the average protein
The rest of the DNA is ______, which interrupt the ______
from Molecular Biology of the Cell
!")
<:&.;.1.;" 1$-$"1 -/9 $:" #"++ #4#+"
Eukaryotic ceII cycIe:
interphase - defined by an absence of ceII division
mitosis
synthesis
!"*
567 ,1 #.;*-#$"9 9%&,/8 #"++ 9,@,1,./
from Molecular Biology of the Cell
!"#+
A&,8,/ .> &"*+,#-$,./B
C:" *.1,$,./ -$ D:,#: $:" 567 :"+,E ,1 .*"/"9
These regions are enriched in AT base pairs. WHY?
Bacteria: One origin of repIication; genome is smaII
enough (~5x10
6
bp) to aIIow repIication every 20-40 min
Eukaryotes: Large genomes, therefore muItipIe origins
of repIication. RepIication forks move ~10 times sIower
(DNA is tightIy packed in chromatin). RepIication from
onIy one fork wouId take ~800 h.
!"##
567 &"*+,#-$,./ F %/D,/9,/8
DNA repIication begins with a partiaI unwinding of the doubIe heIix at
an area known as the replication fork.
DNA poIymerase extends the _____ of a short RNA primer.
This unwinding is accompIished by an enzyme known as DNA helicase.
The unwound section appears under eIectron microscopes as a "bubbIe"
and is thus known as a replication bubble.
animation: http://www.johnkyrk.com/DNAreplication.html
!"#!
567 &"*+,#-$,./ F *&,;,/8
As the two strands separate,
the DNA poIymerase enzyme moves into position...
Short segments of RNA
(RNA primers)
are used as the starting
point for DNA synthesis.
ImportantIy, there is a
leading strand and a
lagging strand.
!"#$
567 &"*+,#-$,./ F 567 *.+4;"&-1"
DNA poIymerase extends from the 3'-end of the primer,
appending new bases toward the 5'-end of the tempIate
The new strand is compIementary and
must be synthesized in the 5' to 3' direction.
DNA poIymerase
cataIyzes the
condensation between
the 5'-phosphate on the
incoming nucIeotide and
the free 3'-OH on the
growing poIynucIeotide
phosphodiester bond
NobeI Prize in PhysioIogy
or Medicine (1959)
to Arthur Kornberg
!"#%
567 &"*+,#-$,./ F +"-9,/8 -/9 +-88,/8 1$&-/91
Because the original DNA strands are complementary and run antiparallel, only one new
strand can begin at the 3' end of the template DNA and grow continuously as the point of
replication (the replication fork) moves along the template DNA.
The result of this side's discontinuous replication is the production of a series of short
sections of new DNA, called ________________ after their Japanese discoverer.
To ensure that this new strand of short segments is made into a continuous strand, the
sections are joined by the action of an enzyme called DNA ligase, which LGATES the
pieces together by forming the missing phosphodiester bond.
!"#&
567 &"*+,#-$,./ F &";.@-+ .> )67 *&,;"&
exonucIease
!"#'
C:" G"/9 &"*+,#-$,./ *&.H+";I
DNA repIication begins in the middIe of a DNA sequence
DNA poIymerase moves on the tempIate in a 3'5' direction
Lagging strand is made by extension
of RNA primers by DNA poIymerase.
RNA primers are removed, DNA gaps
are fiIIed, and DNA fragments are
Iigated together.
PROBLEM: __________
_____________________
_____________________
_____________________
http://www.senescence.info/telomeres.html
Bacteria have circuIar chromosomes,
so this is not a probIem for them
!"#(
C"+.;"&"1
TeIomere: Many tandem repeats of short sequence
(humans ________, extending for ~10,000 nt)
50-100 nt are Iost during each ceII division
After many generations, daughter ceIIs wiII inherit
defective chromosomes; ceIIs wiII die or no Ionger divide
TeIomeres are potentiaI "measuring sticks" for the
ceII to count ceII divisions and to protect against
uncontroIIed growth (cancer)
!"#)
C"+.;"&-1" ,1 -/ "/J4;" $:-$ "E$"/91 $"+.;"&"1
TeIomerase binds
TeIomerase extends
DNA poIymerase
can then prime and
synthesize ends
NobeI Prize in PhysioIogy
or Medicine (2009)
to BIackburn, Greider,
and Szostak
!"#*
C"+.;"&-1" -1 -/ -/$,K#-/#"& $-&8"$
!"!+
)67 14/$:"1,1B C&-/1#&,*$,./
AII ceIIs, from bacteria to humans, foIIow the centraI dogma:
DNA RNA protein
Big difference between prokaryotes and eukaryotes:
RNA splicing occurs onIy in eukaryotes
NobeI Prize in PhysioIogy
or Medicine (1959)
to Severo Ochoa
NobeI Prize in Chemistry
(1959)
to Roger Kornberg
!"!#
)67 14/$:"1,1B C&-/1#&,*$,./
Transcription produces RNA that is
compIementary to one DNA strand
Transcription of RNA proceeds in 5'3' direction
(i.e., DNA information is read in 3'5' direction)
UnIike repIication, the product RNA strand does NOT
remain H-bonded to the DNA tempIate. The RNA is
dispIaced, and the DNA doubIe heIix is formed again.
Three phases in both prokaryotes and eukaryotes:
Initiation, Elongation, Termination
It is important to understand prokaryotic transcription!
!"!!
C&-/1#&,*$,./ ,/ *&.3-&4.$"1B =/,$,-$,./
RNA poIymerase recognizes and binds the promoter DNA
RNA poIymerase can:
- Iink ribonucIeotides
- start an RNA chain without a primer
RNA poIymerase unwinds dsDNA at the promoter site
RNA poIymerase is a muIti-subunit enzyme
( factor "reads" the DNA and enabIes specific binding)
One DNA strand acts as tempIate for a compIementary
ribonucIeotide, and two RNA nt are joined by the RNA poI.
factor is eventuaIIy ejected, and eIongation can proceed
!"!$
Promoters consist of two short sequences:
1. -10 eIement (Pribnow box, TATA box)
2. -35 eIement
Sequences wiII differ sIightIy from gene to gene
Abundant proteins have "stronger" promoters
that enabIe more frequent initiation
Promoter sequences are __________; this determines which
strand is transcribed. The choice of tempIate strand for
each gene is determined by the promoter Iocation and
orientation. Throughout the genome, both strands are used.
C&-/1#&,*$,./ ,/ *&.3-&4.$"1B L&.;.$"&
!"!%
EIongation proceeds at a rate of ~50 nt/s
C&-/1#&,*$,./ ,/ *&.3-&4.$"1B 2+./8-$,./
!"!&
For most genes, the termination sequence has
1. Two-foId symmetric DNA sequence;
when transcribed, the RNA forms a hairpin
2. A string of A-T base pairs
Hairpin structure
forces RNA away from
the poIymerase.
This is enhanced by the
weak A-U bonds at the
DNA-RNA hybrid site
C&-/1#&,*$,./ ,/ *&.3-&4.$"1B C"&;,/-$,./
!"!'
A@"&-++ @,"D .> $&-/1#&,*$,./ ,/ *&.3-&4.$"1
!"!(
A@"&-++ @,"D .> $&-/1#&,*$,./ ,/ *&.3-&4.$"1
!"!)
The DNA does not Ieave the nucIeus,
so transcription happens in the nucIeus.
RNA poIymerase I (PoI I):
transcribes ribosomaI RNA (rRNA)
RNA poIymerase II (PoI II):
transcribes messenger RNA (mRNA)
RNA poIymerase III (PoI III):
transcribes transfer RNA (tRNA)
C&-/1#&,*$,./ ,/ "%3-&4.$"1
!"!*
Promoter is usuaIIy ~50 bp from transcriptionaI start site
Purified PoI II cannot initiate transcription in vitro
Transcriptional activators
are moduIar proteins:
_________________
_________________
C&-/1#&,*$,./ ,/ "%3-&4.$"1B =/,$,-$,./
Transcription factors are required to
position PoI II at promoter
puII apart the DNA strands
reIease PoI II from the promoter after initiation
!"$+
C&-/1#&,*$,./B *&.3-&4.$"1 @"&1%1 "%3-&4.$"1
Prokaryotes
Eukaryotes
!"$#
L.1$K$&-/1#&,*$,./-+ ;.9,>,#-$,./1
Prokaryotes: primary mRNA transcripts are ready for transIation
Eukaryotes: pre-mRNA made in nucIeus; transIation in cytopIasm
post-transcriptionaI processing takes pIace in nucIeus
2. poIy-A taiI added to 3'-end by poIy-A poIymerase
FUNCTION: _________________________________
1. 7-methyIguanosine "cap" added to 5'-end
FUNCTION: __________________________;
!!!!!!!!!!!!!!!!!!!!!!!!!!!!!
3. Introns are spIiced out to form finaI mRNA
!"$!
!"$$
MK;"$:4+8%-/.1,/" #-*
triphosphate bridge
7-methyl-G
!"$%
L.+4K7 $-,+
1. mRNA is cIeaved after AAUAAA site
2. 50-250 adenosine residues are added to 3'-end
through action of poIy-A poIymerase and ATP
!"$&
*&"K;)67 1*+,#,/8
intron
CataIyst: spIiceosome
5'-exon 3'-exon
5' 3'
5' 3'
spIiced exons
different protein
sequence
Pre-mRNA spIicing occurs for
aImost aII eukaryotic genes
NobeI Prize in PhysioIogy
or Medicine (1993)
to Roberts and Sharp
for discovery of spIit genes
!"$'
N%,#3 >-#$1 -H.%$ *&"K;)67 1*+,#,/8
Average # of introns in a human gene: 8
Average Iength of a human gene exon: 150 nucIeotides
Average Iength of a human gene intron: 3500 nucIeotides
Largest intron known: 2,400,000 nucIeotides (muscIe protein gene)
(from Voet & Voet, Biochemistry 3rd ed.)
!"$(
O&.; 8"/" $. *&.$",/
!"$)
P*+,#,/8 ,/#&"-1"1 8"/"$,# 9,@"&1,$4 D,$:.%$
,/#&"-1,/8 $:" /%;H"& .> 8"/"1
AIternative spIicing _________________________!
!"$*
L&.$",/1 14/$:"1,1 Q $&-/1+-$,./
The Genetic Code:
64 codons for 20 amino acids
A codon (tripIet of bases) specifies a given amino acid
!"%+
R.D 8"/"$,# #.9" ,1 &"-9 ,1 @"&4 ,;*.&$-/$
Insertion of one or two nucIeotides is aIso IikeIy
to Iead to an unexpected stop codon.
!"%#
<./@"&1,./ .> #.9./1 $. *&.$",/1 @,- $)67
Structure of a tRNA:
______________ is matched
by ribosome to proper
mRNA codon
________________________
cataIyzes the attachment
between tRNA and amino acids
!"%!
Initiation, EIongation, Termination
Prokaryotic
Ribosome:
16S rRNA recognizes the Shine-Dalgarno sequence
(RBS, ribosome binding site), which is
5-10 bp upstream of the AUG start codon
5'-.AGGAGGUXXXXXXXAUG...3'
3'-.UCCUCCA.-5'
bacteriaI mRNA
16S rRNA
S-D sequence start codon
anti-S-D sequence
50S and 30S
16S, 21 poIypeptides
70S
5S, 23S,
32 poIypeptides
C&-/1+-$,./ ,1 *"&>.&;"9 H4 $:" &,H.1.;"
!"%$
The ribosome is a Iarge compIex of both protein and rRNA
Prokaryotes - transIation occurs on growing mRNA
Eukaryotes - transIation typicaIIy occurs after mRNA
processing and export to the cytopIasm
C&-/1+-$,./ ,1 *"&>.&;"9 H4 $:" &,H.1.;"
NobeI Prize in Chemistry
(2009)
to Ramakrishnan, Steitz,
and Yonath
for ribosome structures
!"%%
A@"&-++ *,#$%&" .> 8"/" "E*&"11,./
!"%&
"A SmaII MoIecuIe TranscriptionaI Activation Domain"
Minter, Brennan & Mapp J. Am. Chem. Soc. 2004, 126, 10504
1) What is the utiIity of an artificiaI transcriptionaI activator?
2) What domains do protein transcriptionaI activators have?
3) Describe the basic approach used in this paper.
4) How were compounds assessed for the desired effect?
5) Others?
L-*"& S T%"1$,./1 >.& 9,1#%11,./ K U
!"%'
"PyrroIysine Encoded by UAG in Archaea: Charging
of a UAG-Decoding SpeciaIized tRNA"
and
"A New UAG-Encoded Residue in the Structure of a
Methanogen MethyItransferase
Science 2002, 296, 1459 and 1462
1) What is the structure of pyrroIysine?
2) Why does it need a speciaIized tRNA?
3) How was it proven that pyrroIysine was incorporated?
4) Others?
L-*"& S T%"1$,./1 >.& 9,1#%11,./ K (