Information Transfer in Cells

Information encoded in a DNA

molecule is transcribed via synthesis
of an RNA molecule
Sequence of the RNA molecule is
"read" and translated into amino acids
in a protein.
Nitrogenous Bases
Pyrimidines
Cytosine DNA! RNA"
#racil RNA"
$hymine DNA"
Purines
Adenine DNA! RNA"
%uanine DNA! RNA"
Properties of Pyrimidines and Purines
&eto'enol tautomerism
Acid(base dissociations
Stron) absorbance of #* li)ht
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Pentoses of Nucleotides
D'ribose RNA"
+'deo,y'D'ribose DNA"
Nucleosides linkage of a base to a sugar
-ase is lin.ed via a )lycosidic bond
$he carbon of the )lycosidic bond is
anomeric
Named by addin) 'idine to the root
name of a pyrimidine or 'osine to the
root name of a purine
Conformation can be syn or anti
Su)ars ma.e nucleosides more /ater'
soluble than free bases
Nucleotides Nucleoside phosphates
&no/ the nomenclature
"Nucleotide phosphate" is redundant0
1ost nucleotides are ribonucleotides
Nucleotides are polyprotic acids
Functions of Nucleotides
Nucleoside 23'triphosphates are
carriers of ener)y e,. A$P! %$P! etc."
-ases serve as reco)nition units
Cyclic nucleotides are si)nal
molecules and re)ulators of cellular
metabolism and reproduction
A$P is central to ener)y metabolism
%$P drives protein synthesis
C$P drives lipid synthesis
#$P drives carbohydrate metabolism
Nucleic Acids Polynucleotides
Polymers lin.ed 43 to 23 by phosphodiester
brid)es
Ribonucleic acid and deo,yribonucleic
acid
&no/ the shorthand notations
Sequence is always read 23 to 43
$his corresponds to "N to C" in proteins
Classes of Nucleic Acids
DNA ' one type! one purpose
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RNA ' 4 or 5" types! 4 or 5" purposes
ribosomal RNA ' the basis of
structure and function of ribosomes
messen)er RNA ' carries the
messa)e
transfer RNA ' carries the amino
acids
The DNA Double eli! stabili"ed by
hydrogen bonds#
"-ase pairs" arise from hydro)en bonds
6r/in Char)a7 had the pairin) data! but
didn3t understand its implications
Rosalind 8ran.lin3s 9'ray :ber di7raction
data /as crucial
8rancis Cric. .ne/ it /as a heli,
;ames <atson :)ured out the ='bonds
The $tructure of DNA an antiparallel double
heli!
Diameter of + nm > len)th of ?.@ million
nm %& coli"
Compact and folded %& coli cell is
+AAA nm lon)"
6u.aryotic DNA /rapped around
histone proteins to form nucleosomes
-ase pairsB A'$! %'C
'essenger (NA transcription product of
DNA
In pro.aryotes! a sin)le mRNA
contains the information for synthesis of
many proteins
In eu.aryotes! a sin)le mRNA codes
for Cust one protein! but structure is
composed of introns and e,ons
%ukaryotic m(NA
DNA is transcribed to produce
hetero)eneous nuclear RNA
mi,ed introns and e,ons /ith
poly A
intron ' intervenin) sequence
e,on ' codin) sequence
poly A tail ' stability
Splicin) produces :nal mRNA /ithout
introns
(ibosomal (NA
Ribosomes are about +(4 RNA! ?(4
protein
rRNA serves as a sca7old for
ribosomal proteins
+4S rRNA in %& coli is the peptidyl
transferase
Transfer (NA
Small polynucleotide chains ' D4 to E5
residues each
Several bases usually methylated
6ach a.a. has at least one unique tRNA
/hich carries the a.a. to the ribosome
43'terminal sequence is al/ays CCA'
a.a.
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Aminoacyl tRNA molecules are the
substrates of protein synthesis
DNA ) (NA Di*erences
+hy does DNA contain thymine,
Cytosine spontaneously deaminates to
form uracil
Repair enFymes reco)niFe these
"mutations" and replace these #s /ith
Cs
-ut ho/ /ould the repair enFymes
distin)uish natural # from mutant #G
Nature solves this dilemma by usin)
thymine 2'methyl'#" in place of uracil
+hy is DNA -./deo!y and (NA is not,
*icinal 'H= )roups +3 and 43" in RNA
ma.e it more susceptible to hydrolysis
DNA! lac.in) +3'H= is more stable
$his ma.es sense ' the )enetic
material must be more stable
RNA is desi)ned to be used and then
bro.en do/n
ydrolysis of Nucleic Acids
RNA is resistant to dilute acid
DNA is depurinated by dilute acid
DNA is not susceptible to base
RNA is hydrolyFed by dilute base
(estriction %n"ymes
-acteria have learned to "restrict" the
possibility of attac. from forei)n DNA
by means of "restriction enFymes"
$ype II and III restriction enFymes
cleave DNA chains at selected sites
6nFymes may reco)niFe 5! @ or more
bases in selectin) sites for cleava)e
An enFyme that reco)niFes a @'base
sequence is a "si,'cutter"
Type II (estriction %n"ymes
No A$P requirement
Reco)nition sites in dsDNA usually
have a +'fold a,is of symmetry
Cleava)e can leave sta))ered or
"stic.y" ends or can produce "bluntI
ends
Names use 4'letter italiciFed codeB
?st letter ' )enusJ +nd!4rd ' species
8ollo/in) letter denotes strain
%coRI is the :rst restriction enFyme
found in the R strain of %& coli
Techniques of Molecular Biology
$he methods depend upon! and /ere
developed from! an understandin) of
the properties of biolo)ical
macromolecules themselves.
=ybridiFation ' the base'pairin)
characteristics of DNA and RNA
DNA clonin) ' DNA polymerase!
restriction endonucleases and DNA
li)ase
PCR ' thermophile DNA polymerase
?. 6lectrophoresis
+. Restriction
4. =ybridiFation
5. DNA Clonin) and )ene e,pression
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2. PCR
@. %enome sequence > analysis
?. %el electrophoresis separates DNA and
RNA molecules accordin) to siFe! shape and
topolo)ical properties.
%el matri, is an inserted! Cello'li.e
porous material that support and
allo/s macromolecules to move
throu)h. A)arose and polyacrylamide
are t/o di7erent )el matrices
DNA and RNA molecules are ne)atively
char)ed! thus move in the )el matri,
to/ard the positive K" pole
Linear DNA molecules are separated
accordin) to siFe
$he mobility of circular DNA molecules is
a7ected by their topolo)ical structures. $he
mobility of the same molecular /ei)ht DNA
molecule /ith di7erent shapes isB
supercoiledM linearM nic.ed or rela,ed
$o
separate DNA of di7erent siFe ran)es
Narro/ siFe ran)e of DNAB use
polyacrylamide
<ide siFe ran)e of DNAB use a)arose
)el
*ery lar)e DNAM4A'2A.b"B use
pulsed':eld )el electrophoresis
NPulsed':eld )el electrophoresis
-S/itchin) bet/een t/o orientationsB the
lar)er the DNA is! the lon)er it ta.es to
reorient
Restriction endonucleases cleave DNA
molecules at particular sites
<hy use endonucleasesG
- $o ma.e lar)e DNA molecules brea. into
mana)eable fra)ments
- Restriction endonucleasesB the nucleases
that cleave DNA at particular sites by the
reco)nition of speci:c sequences
- $he tar)et site reco)niFed by
endonucleases is usually palindromic a
re)ion of DNA in /hich the sequence of
nucleotides is identical /ith an inverted
sequence in the complementary strandB
%AA$$C is a palindrome of C$$AA%."
6ndonucleases are used to ma.e
restriction mapB
e.). the combination of %coRI K indIII
Allo/s di7erent re)ions of one
molecule to be isolate and a )iven
molecule to be identi:ed
A )iven molecule /ill )enerate a
characteristic series of patterns /hen
di)ested /ith a set of di7erent
enFymes
Di7erent enFymes reco)niFe their speci:c
tar)et sites /ith di7erent frequency
6,.
%coRI Reco)niFe he,americ sequenceB
5
'@
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-lunt 6nds
Stic.y 6nds
$au4A? Reco)niFe tetrameric
sequenceB 5
'5
$hus Sau4A? cuts the same DNA
molecule more frequently
$he 2O protrudin) ends of are said to
be Pstic.yI because they readily
anneal throu)h base'pairin) to DNA
molecules cut /ith the same
enFyme
DNA hybridiFation can be used to
identify speci:c DNA molecules
Hybr!"ato#: the process of base'pairin)
bet/een complementary ssDNA and RNA
from t/o di7erent sources
Probe: a labeled! de:ned sequence used to
search mi,tures of nucleic acids for
molecules containin) a complementary
sequence
$a!oact%e label#g: display and(or
ma)nify the si)nals by radioactivity
&o#-ra!oact%e label#g: display and(or
ma)nify the si)nals by anti)en labelin)!
antibody bindin)! enFyme bindin) or
substrate application si)nal release"
'#! label#g: put the labels at the ends
6,. 2O'end labelin)B polynucleotide
.inase PN&"
4O'end labelin)B terminal transferase
(#for) label#g: put the labels internally
&ck tra#slato#: DNase I to introduce
random nic.s DNA pol I to remove
dN1Ps from 4O to 2O and add ne/ dN1P
includin) labeled nucleotide at the 4O
ends.
He*a#ucleot!e pr)ere! label#g:
Denature DNA add random
he,anucleotide primers and DNA pol
synthesis of ne/ strand incorporatin)
labeled nucleotide.
+tra#!-spec,c D&A probes: 6,. 1?4 DNA
as templateJ the missin) strand can be
re'synthesiFed by incorporatin)
radioactive nucleotides
+tra#!-spec,c $&A probes: labeled by
transcription.
+e-ue#c#g
.wo ways for se-ue#c#g:
?. DNA molecules radioactively labeled
at 2O termini" are subCected to 5
re)iments to be bro.en preferentially at
%s! Cs! $s! As! separately.
+. chain'termination methodB ddN$Ps are
chain'terminatin) nucleotidesB the
synthesis of a DNA strand stops /hen a
ddN$P is added to the 4O end
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+out0er# Blot
A#alyss
Date: August
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Group:
.0e s0ortgu# strategy permits a partial
assembly of lar)e )enome sequence
If /e /ant to sequence a much lar)er
and more complicated eu.aryotic
)enome usin) the short)un strate)y.
<hat can /e doG
8irstly! libraries in di7erent level
should be constructed.
$he DNA fra)ment can be easily
e,tracted and sequenced
automatically.
Sophisticated computer pro)rams
have been developed to assemble the
randomiFed DNA fra)ment! formin)
conti)s.
A sin)le conti) is about 2A!AAA to
+AA!AAA bp. ItOs useful to analysis of
fruit Qy )enome that contains an
avera)e of one )ene every ?A.b.
If /e /ant to analyFe human )enome!
conti)s should be assembled into
sca7olds.
.0e pare!-e#! strategy per)ts t0e
asse)bly of large ge#o)e se-ue#ce
$he main limitation to producin) lar)e
conti)s is the occurrence of repetitive
sequence. <hyG"
$o solve this problem! paired'end
sequencin) is developed.
$he same )enomic DNA is also used to
produce recombinant libraries
composed of lar)e fra)ments bet/een
4R?AA.b.
$he end of each clone can be
sequenced easily! and these lar)er
clones can :rstly assemble to)ether.
If a lar)er sca7old is needed! you
should use a clonin) vector that can
carry lar)e DNA fra)ment! at least
?AA.b". -AC is a )ood choice.
Ge#o)e-w!e a#alyss
$he purpose of this analysis is to
predict the codin) sequence and other
functional sequence in the )enome.
8or the )enomes of bacteria and
simple eu.aryotes! :ndin) HR8 is very
simple and e7ective.
8or animal )enomes! a variety of
bioinformatics tools are required to
identify )enes and other functional
fra)ments. -ut the accuracy is lo/.
$he most important method for
validatin) protein codin) re)ions and
identify those missed by current )ene
:nder pro)ram is the use of cDNA
sequence data.
$he mRNAs are :rstly reverse
transcripted into cDNA! and these
cDNA! both full len)th and partial! are
sequenced usin) short)un method.
$hese sequence are used to )enerate
'+. 1e*presse! se-ue#ce tag2
database. And these 6S$s are ali)ned
onto )enomic sca7olds to help us
identify )enes.
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ReferenceB Nucleotides and Nucleic Acids to
accompany -iochemistry! +(e -y Re)inald
%arrett and Charles %risham
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