BIOL 3301 - Genetics Ch13A - Transcription in Prokaryotes St
Transcript of BIOL 3301 - Genetics Ch13A - Transcription in Prokaryotes St
-
8/18/2019 BIOL 3301 - Genetics Ch13A - Transcription in Prokaryotes St
1/49
Gene Expression:
Transcription
The synthesis of an RNA copy of
a segment of DNA
-
8/18/2019 BIOL 3301 - Genetics Ch13A - Transcription in Prokaryotes St
2/49
Gene Expression
• 1909, Archibald Gerrod - alkaptonuria, a
hereditary disease, was caused by the absence
of an enzye that breaks down a specific
substrate, alkapton!
• "he syptos of an inherited disease reflect a
person#s inability to synthesize a particular
enzye
-
8/18/2019 BIOL 3301 - Genetics Ch13A - Transcription in Prokaryotes St
3/49
• 19$0s, Geor%e &eadle and &oris Ephrussi-
each utation affectin% eye color in
Drosophila blocks pi%ent synthesis at a
specific step by pre'entin% production of the
enzye that catalyzes that step!
• (either the cheical reactions nor the
enzyes were known at the tie!
-
8/18/2019 BIOL 3301 - Genetics Ch13A - Transcription in Prokaryotes St
4/49
)ne Gene * )ne Enzye
• &eadle and Edward "atu - etabolis of a
bread old, Neurospora crassa!
•"hey utated Neurospora with +-rays andscreened the sur'i'ors for utants that
differed in their nutritional needs!
• "heir results pro'ided stron% e'idence for the
one gene – one enzyme hypothesis.
-
8/18/2019 BIOL 3301 - Genetics Ch13A - Transcription in Prokaryotes St
5/49
Copyright© 2002 Pearson Education, Inc., publishing as Benjamin Cummings
Fig. 17.1
-
8/18/2019 BIOL 3301 - Genetics Ch13A - Transcription in Prokaryotes St
6/49
Human Diseases Due To
Metabolic Disorders !henyl"etonuria# $:$%#&& chr$%# gene for
phenylalanine hydroxylase
Albinism# $:''#&& tyrosinase
Tay()achs# $:'#*& chr$+# hexA# N(acetylhexaminidase# lysosomal storage disease#
neurological degeneration
,ystic fibrosis# $:%#&&& in ,aucasians# $:$-#&&& in
African(American. $:/&&& in Asians# chr-#
deletion of three nucleotides# ion transport through
membranes is impaired
-
8/18/2019 BIOL 3301 - Genetics Ch13A - Transcription in Prokaryotes St
7/49
(ew entral o%a
• (ot all %enes encode proteins
• (ot all %ene transcripts are translated
•.e'erse transcription can occur
• "here are ore than three types of .(A/
- mRNA
( rRNA ( tRNA
( snRNA 0 small nuclear DNA
-
8/18/2019 BIOL 3301 - Genetics Ch13A - Transcription in Prokaryotes St
8/49
What is a gene?
-
8/18/2019 BIOL 3301 - Genetics Ch13A - Transcription in Prokaryotes St
9/49
A gene is a DNA sequence on a chromosome that
specifies the information that is required by the cell
to make a specific gene product.
What is a gene product?
The DNA sequence of a gene also specifies when a gene product
should be made, in what cells a gene product should be made and how
much of the gene product should be made
DNA (genetic code)
What
Where – tissue specific ( turn on/off)
When
How much -
-
8/18/2019 BIOL 3301 - Genetics Ch13A - Transcription in Prokaryotes St
10/49
-
8/18/2019 BIOL 3301 - Genetics Ch13A - Transcription in Prokaryotes St
11/49
.(A ynthesis
• .(A is synthesized usin% (A as a teplate
• )nly one strand is read * teplate strand andnonteplate strand sense and antisense2
• .e3uireents for transcription/ * .ibonucleoside triphosphate ("4s * (A teplate
* .(A polyerase
• Enzye .(A polyerase catalyzes the process oftranscription
• "ranscription occur unidirectionally, 5# to $#
• )nly a se%ent of a (A is transcribed
-
8/18/2019 BIOL 3301 - Genetics Ch13A - Transcription in Prokaryotes St
12/49
6ost .(A when ade in the cell is ss.(A!
7t#s %eneral cheical structure is 'ery siilar to that of (A, except for the 8#-) and the
use of uracil! (A has a 8#- in deoxyribose and
thyine instead of uracil!
ss.(A is like water in that it will! ! ! :::
-
8/18/2019 BIOL 3301 - Genetics Ch13A - Transcription in Prokaryotes St
13/49
Transcription in E. coli
(in prokaryotes)
• Enzye in'ol'ed in transcription- (A
dependent RNA polymerase
• Rna polymerase activity: soe siilarities,
soe differences to DNA polymerase
activities
-
8/18/2019 BIOL 3301 - Genetics Ch13A - Transcription in Prokaryotes St
14/49
Similarities
• ;ses nucleoside 5
-
8/18/2019 BIOL 3301 - Genetics Ch13A - Transcription in Prokaryotes St
15/49
Differences/
• ;ses ribonucleoside 5
-
8/18/2019 BIOL 3301 - Genetics Ch13A - Transcription in Prokaryotes St
16/49
E. coli RNA polymerase
• =ery lar%e protein coplex, consists of fi'e subunits!
• 8 identical alpha subunits and 1 each of beta, beta, andsi!ma"
• "he si%a subunit dissociates fro the enzye easily -lea'es shortly followin% initiation, critical for reco%nitionof start of %ene!
• holoen#yme - coplete enzye - all fi'e subunitsto%ether but basic polyerization reaction possible without
si%a subunit -• $ore en#yme/ 8 alpha, 1 beta and 1 beta
-
8/18/2019 BIOL 3301 - Genetics Ch13A - Transcription in Prokaryotes St
17/49
.(A 4olyerase
• ore .(A 4olyerase
>>>>>>>>>>>>>>>>>
* &inds (A
* atalyzes .(A synthesis but with no specificity
* annot reco%nize
prooter se3uence
• oloenzye .(A4
>>>>>>>>>>>>>>>>>
- ? reduces affinity to
nonspecific (A- Greatly increases affinity
to prooter
- Ali%ns .(A4 with
transcription site @12
-
8/18/2019 BIOL 3301 - Genetics Ch13A - Transcription in Prokaryotes St
18/49
RNA% &oloen#yme
-
8/18/2019 BIOL 3301 - Genetics Ch13A - Transcription in Prokaryotes St
19/49
'r!ani#ation of control re!ions
• "he base where transcription starts is nubered
• &ases distal of the @1 base fro the codin% re%ion are referred
to as upstream and are nuber as/ -1, -8, -$ !!!
• "hose bases adacent to the @1 base close to the codin% re%ionare referred to as do*nstream and are positi'ely nubered!
• "he re%ions of (A that bind .(A polyerase are called
promoters"
• "he re%ions of (A that bind re%ulatory proteins are calledoperators!
-
8/18/2019 BIOL 3301 - Genetics Ch13A - Transcription in Prokaryotes St
20/49
-
8/18/2019 BIOL 3301 - Genetics Ch13A - Transcription in Prokaryotes St
21/49
4rooters
• E. coli prooters contain conser'ed (A
se3uences consensus se3uences2/
* "A"AA", 10 nucleotides upstrea if initiation
site - 10 re%ion2, 4ribnow box
* ""GAA, - $5 re%ion
* cis-actin% eleents next to or on the sae
side2
-
8/18/2019 BIOL 3301 - Genetics Ch13A - Transcription in Prokaryotes St
22/49
Alignment of multiple
promoters
-
8/18/2019 BIOL 3301 - Genetics Ch13A - Transcription in Prokaryotes St
23/49
4rooters
• A se3uence upstrea of the start of the .(Acodin% se3uence
• Generally are found at -$5 and -10 positions
• onsensus se3uence for -$5 re%ion is 5#-""GAA-$#
• onsensus se3uence for -10 position is 5# *
"A"AA"-$#• "he ore siilarity to the consensus
se3uence, the stron%er the prooter
-
8/18/2019 BIOL 3301 - Genetics Ch13A - Transcription in Prokaryotes St
24/49
'r!ani#ation 'f %romoter Re!ion +n ac- 'peron
-
8/18/2019 BIOL 3301 - Genetics Ch13A - Transcription in Prokaryotes St
25/49
%romoters
• "he .(A polyerase core uses the -10 se3uence
"A"AA"2 to bind and orient where to be%in
transcription!• "he .(A polyerase si%a subunit uses the -$5 and
-10 to soe de%ree2 se3uence to bind and stabilize
the polyerase coplex!
-
8/18/2019 BIOL 3301 - Genetics Ch13A - Transcription in Prokaryotes St
26/49
4rooters
• ontrol the efficiency of transcription
• tron% prooters * initiation e'ery 1-8
seconds
• Beak prooters * initiation e'ery 10-80
inutes
• 6utations of the prooter re%ion ay reduceor enhance transcription
-
8/18/2019 BIOL 3301 - Genetics Ch13A - Transcription in Prokaryotes St
27/49
4rooters
• ifferent si%a subunits can direct the polyerase to
specific %enes! ynthesis of new or uni3ue si%a
subunits can redirect the polyerase to transcribe a
new set of %enes• "he stren%th of a prooter is related to its identity to
the consensus se3uence
• (ot all %enes ha'e the -10 and -$5 se3uence in the
prooter 2
-
8/18/2019 BIOL 3301 - Genetics Ch13A - Transcription in Prokaryotes St
28/49
Enhancers
• 4ositi'e re%ulator proteins often interact withthe .(A polyerase and stabilize it at poor prooters that do not closely atch the
consensus se3uence• Enhancers can accelerate the con'ersion of
closed coplexes into open coplexes
• 6any bacterial responses are controlled bytwo-coponent re%ulatory systes
-
8/18/2019 BIOL 3301 - Genetics Ch13A - Transcription in Prokaryotes St
29/49
"ranscription in 4rokaryotes
• "hree sta%es/
* initiation
* elon%ation
* terination
-
8/18/2019 BIOL 3301 - Genetics Ch13A - Transcription in Prokaryotes St
30/49
7nitiation of "ranscription 7n
4rokaryotes
• .(A polyerase for a holoenzye - core enzye
subunits two C, one D and one D# bound by si%a
factor ?2
• oloenzye binds to the prooter in two steps/ * &inds loosely to -$5 box closed prooter coplex2
* &inds ore ti%htly to -10 box, accopanied by untwistin%
of (A open prooter coplex2
• &indin% to a prooter allow correct orientation of the
holoenzye
-
8/18/2019 BIOL 3301 - Genetics Ch13A - Transcription in Prokaryotes St
31/49
ow does the polyerase know
what direction to %o in:
-
8/18/2019 BIOL 3301 - Genetics Ch13A - Transcription in Prokaryotes St
32/49
Specific bindin! of RNA
polymerase • .(A polyerase can bind to double-stranded (A
nonspecifically with low affinity >>>>>>>>>>>>>>>>>>>
• "he holoenzye with si%a subunit2 binds specifically to
both the -10 and -$5 se3uence with hi%h affinity ---- resultin%
in a closed.promoter comple/
• "he holoenzye unwinds about 15 bases in double-stranded
(A around the initiation site for transcription ---- resultin% in
an open. promoter comple/
• "ranscription starts by base pairin% of two r("4s that are oined
-
8/18/2019 BIOL 3301 - Genetics Ch13A - Transcription in Prokaryotes St
33/49
7nitiation of "ranscription 7n
4rokaryotes
• i%a ?2 factor * se'eral types
* Allow the holoenzye to reco%nize "A"A box
* "A"AA" se3uence is reco%nized by si%a factor ?E0 , protein 0,000a
-
8/18/2019 BIOL 3301 - Genetics Ch13A - Transcription in Prokaryotes St
34/49
-
8/18/2019 BIOL 3301 - Genetics Ch13A - Transcription in Prokaryotes St
35/49
7nitiation
• .(A polyerase catalyzes the insertion of the
first 5#-ribonucleoside phosphate,
copleentary to the first nucleotide at the
start site
• (o prier is re3uired
• 4rocess continues in 5# to $# direction
-
8/18/2019 BIOL 3301 - Genetics Ch13A - Transcription in Prokaryotes St
36/49
-
8/18/2019 BIOL 3301 - Genetics Ch13A - Transcription in Prokaryotes St
37/49
Elon%ation of "ranscription 7n
4rokaryotes
• (A helix unwinds to for transcription bubble
• About 10 nt are synthesized
• ? factor separates fro holoenzye
• "he core enzye copletes the transcription of the
%ene
• As .(A polyerase o'es, it untwists (A ahead
and reanneal (A behind it• "ranscription proceeds at a speed of $0-50 ntFsec
• .(A polyerase has two proofreadin% acti'ities
-
8/18/2019 BIOL 3301 - Genetics Ch13A - Transcription in Prokaryotes St
38/49
5’-TTTTTTTTTTTTTTTTTTTT-3’
3’-AAAAAAAAAAAAAAAAA-5’
Gene sequences can be encoded on either strand of
the double helix.
DNA double helix
What will the RNA sequence be of the geneencoded on the top strand?
-
8/18/2019 BIOL 3301 - Genetics Ch13A - Transcription in Prokaryotes St
39/49
5’-TTTTTTTTTTTTTTTTTTTT-3’
3’-AAAAAAAAAAAAAAAAA-5’
Gene sequences can be encoded on either strand of
the double helix.
DNA double helix
transcription of the gene on the top strandRNA polymerase uses the bottom strand
as a template to make RNA.
5’-UUUUUUUUUUUUUUUU-3’
-
8/18/2019 BIOL 3301 - Genetics Ch13A - Transcription in Prokaryotes St
40/49
5’-TTTTTTTTTTTTTTTTTTTT-3’
3’-AAAAAAAAAAAAAAAAA-5’
Gene sequences can be encoded on either strand of
the double helix.
DNA double helix
transcription of the gene on thebottom strand
RNA polymerase uses the top strand as a
template to make RNA.
-
8/18/2019 BIOL 3301 - Genetics Ch13A - Transcription in Prokaryotes St
41/49
Elongation
Template strand 3'4 to +4 DNA5 0 RNA
strand +4 to '4# complementary 3sense
RNA5
Nontemplate strand# +4 to '4 DNA# RNA
se6uence is identical 3antisense RNA5
-
8/18/2019 BIOL 3301 - Genetics Ch13A - Transcription in Prokaryotes St
42/49
0lon!ation:
• "he core enzye lea'es the prooter site
• "he core enzye tra'els alon% (A teplate
• "he (A is unwound in front and is rewound behind so that approxiately 1 base pairs are
always unwound
•r("4s are added to %rowin% .(A 'ia base pairin% and phosphodiester bond foration
-
8/18/2019 BIOL 3301 - Genetics Ch13A - Transcription in Prokaryotes St
43/49
Termination:
• "he core enzye encounters a terination
si%nal
• .(A is released fro (A teplate and fro
the enzye
• .(A polyerase dissociates fro (A
-
8/18/2019 BIOL 3301 - Genetics Ch13A - Transcription in Prokaryotes St
44/49
"erination
• Enzye reaches a specific nucleotidese3uence that acts as a terination si%nal
• "erination si%nal is about
>>>>>>>>>>>>>>>>>>>>> in len%th• "he se3uence is actually transcribed into .(A
• (ewly fored transcript folds back on itself
forin% >>>>>>>>>>>>>>>>>>>>> secondarystructure
-
8/18/2019 BIOL 3301 - Genetics Ch13A - Transcription in Prokaryotes St
45/49
"erination
• oeties a terination factor rho a lar%e
hexaeric protein2 facilitates the terination
• Enzye stops addin% nucleotides
• .(A transcript is released fro teplate
• ore polyerase enzye dissociates
-
8/18/2019 BIOL 3301 - Genetics Ch13A - Transcription in Prokaryotes St
46/49
-
8/18/2019 BIOL 3301 - Genetics Ch13A - Transcription in Prokaryotes St
47/49
4olycistronic "ranscription
• 4olycistronic %enes
;ni3ue to prokaryotes
•6ore than one polypeptide is encoded by asin%le strand of .(A
• pacers after stop codons2 separate the
se3uences so that indi'idual polypeptides can
be translated as sin%le entities
-
8/18/2019 BIOL 3301 - Genetics Ch13A - Transcription in Prokaryotes St
48/49
4olycistronic "ranscription
" i i A d " l i 7
-
8/18/2019 BIOL 3301 - Genetics Ch13A - Transcription in Prokaryotes St
49/49
"ranscription And "ranslation 7n
4rokaryotes• 4rokaryotic (A is located in cytoplas and
ribosoes are distributed in the cytoplas
• Allows for siultaneous occurrence of/
* "ransciption of (A to .(A
* "ranslation of that .(A to proteins