Molecular Biology
Molecular Biology
Molecular Biology
Complementarity of nucleotide allo! eac" trand to act a a template# or mold# for t"e ynt"e i of a ne! complementary trand
T"i feat i performed $y a clu ter of protein t"at to%et"er form a replication machine& Becau e eac" parental trand er'e a t"e template for one of the original (old) trand plus one strand that is completely tyle of replication i aid to $e semiconservative *o! +e ,no!- ./e el o"n-Sta"l e0periment
ne! trand# eac" of t"e dau%"ter DNA dou$le "elice end up with one
new; t"i
of
22222222222222222222222222222222222222222222222222222222222222 2222222222222222
initiator proteins
t"at bind
to t"e DNA and pry the two strands apart# brea#ing the hydrogen bonds
Alt"ou%" t"e "ydro%en $ond collecti'ely ma3e t"e DNA "eli0 'ery ta$le#
$eparating a short length of DNA a fe! $a e pair at a time t"erefore doe not re%uire a large energy input# !"ic" occur !it" t"e a i tance of t"e e protein at normal temperature & T"e po ition at !"ic" t"e DNA i fir t opened are called replication
22222222222222222222222222222222222222222222222222222222222222 2222222222222222
circular DNA
Be%innin% DNA replication at many place at once %reatly shortens the time a cell need to copy it
entire %enome&
5Once an initiator protein $ind to DNA at t"e replication ori%in and Locally open up t"e dou$le "eli0# it attracts a group of proteins t"at carry out DNA replication& T"e e protein form a
pecific function&6
$idirectional&
DNA
polymera e#
Nucleotide enter t"e reaction initially a nucleo ide triphosphates!"ic" pro'ide t"e ener%y for polymeri8ation& DNA i ynt"e i8ed in t"e
01(to(21
of nucleotide to t"e
phosphodiester $ond $et!een t"i end and t"e 9:-p"o p"ate %roup of t"e
incomin% nucleotide of a polynucleotide c"ain i t"e fundamental reaction $y !"ic" DNa i ynt"e i8ed& 22222222222222222222222222222222222222222222222222222222222222 2222222222222222 3!. I$ T!E )*' $.*T!E$I$ )O*E I*
01(TO(21
)IRECTIO* 4after
all the same phosphodiester bond is made between the same #ind of molecules and the entire reaction scenario is the same5"""""""" A
t"e
01(to(21
direction&
PLEASE /A,E S;RE <O; DRA+ O;T A S,ETC* O7 T*E *<POT*ESES INCL;DING T*E ACTI=ATED N;CLEOTIDES (A) In t"e "ypot"etical
21(to(01
remo'e an incorrect nucleotide# !"ic" !ould t"en $loc3 addition of t"e correct nucleotide and t"ere$y prevent further chain elongation& REACTION
DOES NOT PROCEED# AS NO *IG*-ENERG< BOND +O;LD BE CLEA=ED (B) Gro!t" in t"e
01(to(21
elon%ated !"en an incorrect nucleotide "a $een added and t"en remo'ed $y proofreadin%
*IG*-ENERG< BOND IS CLEA=ED# PRO=IDING T*E ENERG< 7OR POL</ERI>ATION 22222222222222222222222222222222222222222222222222222222222222 2222222222222222
nucleotide in a DNA dou$le "eli0# it cannot start a completely ne! DNA trand& A different en8yme i needed?one t"at can $e%in a ne! polynucleotide c"ain
imply $y @oinin% t!o nucleotide to%et"er !it"out t"e need for a $a e-paired end& This en6yme doe not# ynt"e i8e DNA& It ma3e a short length of7R*' (ri$onucleic acid)?u in% t"e DNA trand a a template& T"i "ort len%t" of RNA# a$out AB nucleotide lon%# i $a e-paired to t"e template trand and provides a base(paired 21 end as a starting
point for )*' polymerase& It t"u er'e a a primer for DNA ynt"e i & T"i en8yme !"ic" i a 3ind of R*' polymerase# i called a PRI8'$E& 7or the leading strand# an R*' primer is needed only once to tart replication at a replication ori%inC once a replication for3 "a $een e ta$li "ed# t"e DNA polymera e i continuou ly pre ented !it" a $a e-paired DE end& But on the lagging trand# !"ere DNA ynt"e i i di continuou # new primers tretc" of unpaired $a e # all t"e time& To produce a continuou ne! DNA trand from t"e many eparate piece of nucleic acid made on t"e la%%in% trand# t"ree additional en8yme are needed to1
are needed continually a t"e mo'ement of t"e replication for3 e0po e a ne!
remo'e t"e RNA primer# (nuclease5 replace it !it" DNA#( repair(dna polymerase) (u
of t"e ad@acent O3a8a3i fra%ment a a primer)
t"e 9Ep"o p"ate end of one ne! DNA fra%ment to t"e ad@acent DE "ydro0yl end of t"e ne0t
Primase can $e%in ne! polynucleotide c"ain # $ut t"i acti'ity i po i$le
$ecau e t"e en8yme does not have proof reading activity& But !"o care Gt"e mi ta3e !ill $e corrected a t"e dna repair-polymera e replace t"e primer &
Protein at a Replication 7or3 Cooperate to 7orm a Replication /ac"ine 7or DNA ynt"e i to proceed# the double heli must be un6ipped a"ead
of t"e replication for3 o t"at t"e incomin% deo0yri$onucleo ide trip"o p"ate can form $a e pair !it" t"e template trand& T!o type of replication protein ?)*' helicases and in%le- trand $indin% protein ?$$9s carry out t"i ta 3 "elica e# u e t"e ener%y of ATP "ydroly i to pry apart t"e dou$le "eli0 a it peed alon% t"e DNA T"e in%le- trand $indin% protein clin% to t"e in%le- tranded DNA e0po ed $y t"e "elica e# tran iently pre'entin% it from re-formin% $a e pair and 3eepin% it in an elon%ated form o t"at it can readily er'e a a template for DNA polymera e&
down the R*' primer needed to start the O#a6a#i fragment at t"e
'ery tip of t"e linear DNA molecule& +it"out a trate%y to deal !it" t"i pro$lem# ome DNA !ill ine'ita$ly $e lo t from t"e end of a DNA molecule eac" time it i replicated& Bacteria ol'e t"i Hend-replicationE pro$lem $y "a'in% circular DNA molecule a c"romo ome & Eu3aryote ol'e it $y "a'in% pecial nucleotide eIuence at t"e end of t"eir c"romo ome !"ic" are incorporated into telomere & T"e e telomeric DNA eIuence attract an en8yme called telomera e to t"e c"romo ome& ; in% an RNA template t"at i part of t"e en8yme it elf# telomerase replenishes the nucleotides that are lost eac" time a eucaryotic c"romo ome i duplicated $y
addin% multiple copie of t"e ame "ort DNA eIuence to t"e c"romo ome end & This e tended- repetitive )*' se%uence then acts as a template that allows replication of the lagging strand to be completed by conventional )*' replication
In tep A# a trNa carryin% t"e ne0t amino acid in t"e c"ain $ind to t"e 'acant a- ite on t"e ri$o ome $y formin% $a e pair !it" t"e codon t"at i e0po ed t"ere& Becau e only one of t"e many type of trNa molecule in a cell can $a e-pair !it" eac" codon# t"i codon determine t"e pecific amino acid to $e added to t"e %ro!in% polypeptide c"ain& t"e aand p- ite are ufficiently clo e to%et"er t"at t"eir t!o trNa molecule are forced to form $a e pair !it" codon t"at are conti%uou # !it" no tray $a e in $et!een& t"i po itionin% of t"e trNa en ure t"at t"e correct readin% frame !ill $e pre er'ed t"rou%"out t"e ynt"e i of t"e protein& In tep 4# t"e car$o0yl end of t"e polypeptide c"ain i uncoupled from t"e trNa at t"e p- ite and @oined $y a peptide $ond to t"e free amino %roup of t"e amino acid lin3ed to t"e trNa at t"e a- ite& t"i reaction i cataly8ed $y an en8ymatic ite in t"e lar%e u$unit& In tep D# a "ift of t"e lar%e u$unit relati'e to t"e mall u$unit mo'e t"e t!o trNa into t"e e- and p- ite of t"e lar%e u$unit& In tep J# t"e mall u$unit mo'e e0actly t"ree nucleotide alon% t"e mrNa molecule# $rin%in% it $ac3 to it ori%inal po ition relati'e to t"e lar%e u$unit& t"i mo'ement re et t"e ri$o ome !it" an empty a- ite o t"at t"e ne0t aminoacyl-trNa molecule can $ind (/o'ie K&L)& a indicated# t"e mrNa i tran lated in t"e 9:-to-D: direction# and t"e N-terminal end of a protein i made fir t# !it" eac" cycle addin% one amino acid to t"e C-terminu of t"e polypeptide c"ain&
E:'8PLE;+<
the tryptophan(repressor
7or e0ample# in E& coli# fi'e %ene code for en8yme t"at manufacture t"e amino acid tryptophan& T"e e %ene are arran%ed in a clu ter on t"e c"romo ome and are tran cri$ed from a in%le promoter a one lon% mRNA molecule from !"ic" t"e fi'e protein are tran lated +"en tryptop"an i pre ent in t"e urroundin% and enter t"e $acterial cell# t"e e en8yme are no lon%er needed and t"eir production i "ut off& T"i ituation ari e # for e0ample# !"en t"e $acterium i in t"e %ut of a mammal t"at "a @u t eaten a meal ric" in protein& T"e e fi'e coordinately e0pre ed %ene are part of an
OPERO*?a set of genes t"at are tran cri$ed into a single mR*'&
Operons are common in $acteria $ut are not found in eucaryotes# !"ere %ene are tran cri$ed and re%ulated indi'idually
in len%t") called the operator# t"at i reco%ni8ed $y a protein called transcription regulator&
t"e tran cription of t"e operon and production of t"e tryptop"an-producin% en8yme &
T"e transcription regulator Mrepressor 4meaning< it represses =switches the gene off in its active form5 i controlled in an in%eniou !ay1 t"e repre or can $ind to DNA only if it "a al o $ound e'eral molecule of tryptop"an
NNNT"e tryptop"an repre or i an allosteric protein< t"e $indin% of tryptop"an cau e a u$tle c"an%e in it t"ree-dimen ional tructure o t"at t"e protein can $ind to t"e operator eIuence&
+"en t"e concentration of free tryptophan in t"e cell drop # t"e repre or no lon%er $ind tryptop"an and t"u no lon%er $ind to DNA# and t"e tryptop"an operon i tran cri$ed& T"e repressor switches production of a et of $io ynt"etic en8yme on and off according to the availability of t"e end product of t"e pat"!ay t"at t"e en8yme cataly8e (a ort of negative feedbac#5
E:'8PLE;><
the C'P(activator
T"e $acterial activator protein C'P "a to $ind cyclic '8P (cA/P) $efore it can $ind to DNA& Gene acti'ated $y CAP are switched on in re pon e to an increa e in intracellular c'8P concentration# !"ic"
i%nal to t"e $acterium t"at %luco e# it preferred car$on ource# i no lon%er a'aila$leC
a a re ult# CAP dri'e t"e production of en8yme capa$le of de%radin% ot"er u%ar &
of the R*'
E:'8PLE;2<
the lac(operon
di acc"aride lactose&
In t"e ABSENCE O7 GL;COSE# C'P switches on %ene t"at let t"e cell to utili6e (meanin%1 tran porter protein # di%e ti'e en8yme etc&&) alternati'e ource of car$on?includin% lacto e& It !ould $e !a teful# "o!e'er# for CAP to induce e0pre ion of t"e lac operon !"en lacto e i a$ ent& T"u t"e Lac repressor shuts off t"e operon in t"e ABSENCE O7 LACTOSE& T"i arran%ement ena$le t"e control re%ion of t"e Lac operon to inte%rate t!o different i%nal # o t"at t"e operon i "i%"ly e0pre ed only !"en t!o condition are met1 o lactose must be present and o glucose must be absent
NNT"i %enetic circuit t"u $e"a'e li3e a !itc" t"at carrie out a lo%ic operation in a computer& +"en lacto e i pre ent AND %luco e i a$ ent# t"e cell e0ecute t"e appropriate pro%ram1 in t"i ca e# tran cription of t"e %ene t"at permit t"e upta3e and utili8ation of lacto e& T"e ele%ant lo%ic of t"e Lac operon fir t attracted t"e attention of $iolo%i t more t"an 9B year a%o& T"e molecular $a i of t"e !itc" !a unco'ered $y a com$ination of %enetic and $ioc"emi try# pro'idin% t"e fir t in i%"t into "o! %ene e0pre ion i controlled& In a eu3aryotic cell# imilar %ene re%ulatory de'ice are com$ined to %enerate increa in%ly comple0 circuit & Indeed# t"e de'elopmental pro%ram
t"at ta3e a fertili8ed e%% to adult"ood can $e 'ie!ed a an e0ceedin%ly comple0 circuit compo ed of imple component li3e t"o e t"at control t"e Lac and tryptop"an operon &
R*' interference
Cellular mec"ani m to top t"e tran lation of a particular m RNA into protein1 t"i i a type of %ene re%ulation po t- tran cription Can occur in D different cenario 1
Cell !ant to control one of it own m R*' from tran latin% Cell !ant to c"ec3 on transposable elements Cell !ant to de troy mal protein formin% m R*'s li3e t"o e of viruses
8icro R*'s 4a class of non coding m R*'s5 are transcribed and processed into double stranded ds R*'s )s R*'s are cut into small segments of ds R*'s by an en6yme )ICER These are called si R*'s 4small interfering R*'s5 $i R*'s are recogni6ed by a protein comple called RI$C 4rna induced silencing
comple 5 which slices the ds rna into two separate strands& It destroys one of it and patrols the cell with the other half to find a complementary R*'& Once it finds the complmentary R*'- IT )E$TRO.$ IT&