biol 3301 - genetics ch13a - transcription in prokaryotes st

8/18/2019 BIOL 3301 - Genetics Ch13A - Transcription in Prokaryotes St

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Gene Expression:

Transcription

The synthesis of an RNA copy of

a segment of DNA


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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


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• 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!


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)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.


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Copyright© 2002 Pearson Education, Inc., publishing as Benjamin Cummings

Fig. 17.1


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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


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(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


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What is a gene?


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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 -


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.(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


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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! ! ! :::


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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


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Similarities

• ;ses nucleoside 5


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Differences/

• ;ses ribonucleoside 5


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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


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.(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


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RNA% &oloen#yme


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'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!


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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


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Alignment of multiple

promoters


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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


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'r!ani#ation 'f %romoter Re!ion +n ac- 'peron


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%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!


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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


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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


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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


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"ranscription in 4rokaryotes

• "hree sta%es/

* initiation

* elon%ation

* terination


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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


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ow does the polyerase know

what direction to %o in:


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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


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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


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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


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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


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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?


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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’


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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.


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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


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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


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Termination:

• "he core enzye encounters a terination

si%nal

• .(A is released fro (A teplate and fro

the enzye

• .(A polyerase dissociates fro (A


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"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


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"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


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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


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4olycistronic "ranscription

" i i A d " l i 7


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"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

biol 3301 - genetics ch13a - transcription in prokaryotes st

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