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TRANSCRIPTION

� Mechanistically similar DNA replication

in terms of substrates and template-

directed growth

� 2 major difference:

y Only 1 DNA template strand is transcribed

for a particular gene

y Only a small fraction of the entire geneticpotential of an organism is relaized in one

cell.



EXISTENCE OF mRNA

� Until about 1960 it was thought that

rRNA represented the set of templates

fro protein synthesis

� Jacob and Monod predicted the

existence of mRNA by analyzing E.coli

mutants that are altered in the control of

lactose metabolism� Operon model

� T2 and T4 barcteriophage



CHARACTERISTICS OF mRNA

� There is a high rate of mRNA synthesisfollowed by rapid degradation

� Because of the rapid synthesis and

degradation, mRNA accumulates rapidlybut not to high steady state levels

� mRNA, being a copy of 2 or morecontiguous genes, is large andheterogenous

� mRNA nucleotide sequence is identicalto one of the template strand of DNA



RNA POLYMERASE

� Polynucleotide phosphorylase was initiallythought to be the RNA-synthesizingenzyme

� Ultimately it turned out that it participates inthe degradation of bacterial mRNA

� In prokaryotes, a single RNA polymerasecatalyzes the synthesis of all 3 RNA

classes� In eukaryotes, 3 classes of polymerase

catalyze the different types of RNA



RNA POLYMERASE

� The 3 classes of RNA Polymerase differ

in their sensitivity to inhibition by -

amanitin

� 2 more inhibitors were used to confirm

other characteristics of transcription

y Cordycepin

Transcription chain terminator y Actinomycin D

Intercalating agent



RNA POLYMERASE KINETICSDNA POLYMERASE RNA POLYMERASE

Turnover number 500 -1000

nucleotides/sec

50 nucleotides/ sec

Number of enzyme/

cell

10 molecules 3,000 molecules

Processivity High High

Accuracy 10-6 10-5

Error ± correction

mechanism

3¶ ±exonuclease GreA and Gre B



RNA POLYMERASE STRUCTURE

SUBUNIT Mr # per enzyme

molecule

Function

36,500 2 Chain initiation , interaction with regulatory

proteins and upstream promoter elements

151,00

0

1 Chain initiation and elongation

¶ 155,000

1 DNA binding

70,000 1 Promoter recognition

11,000 1 unknown



RNA POLYMERASE STRUCTURE



INITIATION OF TRANSCRIPTION



ELONGATION: Incorporation of

Ribonucleotides



PROMOTER RECOGNITION

� The most frequently transcribed genes

initiate transcription about once every

10sec,whereas some genes are

transcribed as infrequently as once per

generation (30-60 min)

� Rate ±limiting step

� Each gene transcribed in E.coli shared ashort adenine and thymine rich

sequence (CONSENSUS SEQUENCE)



CONSERVED SEQUENCES IN

PROMOTERS



TERMINATION

� In bacteria, 2 distinct types of

termination events exists:

y Factor ±Independent

y Factor ± Dependent



FACTOR-INDEPENDENT

� 2 structural

features:

y The symmetrical

GC-rich segmentsthat in the trasncript

have the potential to

form a stem-loop

structure

y A downstream run

of four to eight A

residues



FACTOR DEPENDENT

TERMINATION

� Less frequent

� Rho protein is ahexamer composed

of identical subunits� It is an RNA ±DNA

helicase whichcontains nucleotide

triphosphataseacivity that isactivated by bindingto polynucleotides



REGULATION OF TRANSCRIPTION

� Most genes are kept in a turned-off until

their products are needed

� Genes concerned primarily with

anabolism are kept turned-on unless the

product of the anabolic sequence is

present, then the genes are repressed



PROKARYOTIC REGULATION OF

TRANSCRIPTION

� The Lactose Operon

� Bacteriophage

�

The SOS regulon� Biosynthetic operons



THE LACTOSE OPERON

� Consists of 3 linked structural genes that

encode enzymes of lactose utilization

plus adjacent regulatory sites

� Structural genes z,y and a encode -

galctosidase, - galactoside permease

and thiogalactoside transacetylase

� In the presence of an inducer, all 3enzymes accumulate simultaneously but

at different levels





REGULATION IN LAC OPERON

� 2 distinct mutant phenotypes:

y Constitutive

y Noninducible

� These 2 mutations mapped in 2 sited o

and i

� Transcription of the 3 structural genes is

initiated near an adjacent site called theoperator

� This will yield a polycystronic mRNA



REGULATION IN LAC OPERON



ISOLATION AND PROPERTIES OF

REPRESSOR

� Isolated by W.Gilbert andB.Muller-Hill in

1966� A tetramer, each

with 360 aminoacids

�

i gene is expressedat a very low rate(10 molecules /cell)but very efficient



THE REPRESSOR BINDING SITE

� The operator is composed of 35 bp, with

28 bp of symmetrical sequence





REGULATION BY GLUCOSE

� Glucose repression or catabolite

repression

� Decrease in the levels of glucose would

increase the cAMP levels

� The increase would trigger activation of

the lac operon by its interaction with a

protein cAMP receptor protein (CRP)





BACTERIOPHAGE



BACTERIOPAHGE

� 2 repressors: cI and Cro

� Each binds 2 different operators.

�

The operators contain 3 repressor binding sites and promoter sites

interspersed with the repressor binding

site

� Transcrition from the 2 promoter-operator sites takes place in opposite

directions along the genome





Lambda cI REPRESSOR

� A dimeric protein with

a subunit molecular

weight of 27,000

� Transcription from

OLPL andORPR is

controlled by cI and

Cro



GENES in LAMBDA PHAGE

� cI and cro ± code for the repressors

� cII and cIII ± stimulate cI synthesis

�

rex ± unknown function� O and P ± initiation of DNA replication

� N ± product interacts with NusA to

prevent termination

� Q ± product activates late gene

transcription



INTERACTION BETWEEN THE 2

REPRESSORS

� OLPL controls transcription of N through

interaction of its repressor binding site

with the cI protein

� However, most regulatory action occurs

at ORPR and it is here that the decision

is made between lysogenic and lytic

infection





Cro REPRESSOR

� Homodimer of 66-residue subunits

folded into 3 alpha-helical regions and 3

beta strands

� Helices 2 and 3 fit into the major groove

of the DNA



THE SOS REGULON

� REGULON ± set of unlinked genesregulated by a common mechanism

� Lytic infection is induced through DNAdamaging treatments (i.e. UV irradiation)

� lexA and recA gene products are thecontrol elements in E.coli SOS regulon

� RecA ± stimulates strand pairing duringrecombination and can stimulate proteolytic

cleavage of proteins when bound to ssDNA� LexA ± a repressor that binds at least 25

different operators scattered in the genome



THE SOS REGULON

� In healthy cells, lexA and recA areexpressed at low levels

� The triger that activates the SOS systemafter damage is ssDNA

� RecA binding within a gap activates LexAproteolysis

� Decrease in conc of LexA removes LexAbarrier to recA transcription. RecA

accumulates� Simultaneously, cleavage of LexA protein

activates transcriptio of all genes under lexA control (including cI repressor)



BIOSYNTHETIC OPERONS

� Since biosynthetic pathways utilize

energy, the regulatory goal is to repress

gene activity by turning off the synthesis

of the enzymes in the pathway when theend product is available

� t rp operon







POSTTRANSCRIPTIONAL

PROCESSING

� The major postranscritional event in

metabolism of prokaryotic mRNA is its

own degradation

� mRNA have half-lives of ~2-3 mins

� Degradation starts from the 5¶ end



POSTTRANSCRIPTIONAL

PROCESSING

� rRNA processing

y Contains 7 different operons for rRNA

species .

y Each one encodes one copy of 16S, 23Sand 5S in a single transcript

y It also includes sequences for 1-4 tRNA

molecules

y Initial transcript form each operon is a 30S

RNA molecule

y RNaseIII



POSTTRANSCRIPTIONAL

PROCESSING

� tRNA processing

y tRNAs are

synthesized in

transcriptscontaining 1-7

tRNAs each all

surrounded by

lengthy flankingsequences



TRANSCRIPTION IN EUKARYOTIC

CELLS

� Transcription is precisely programmed

during development and tissue

differentiation

� Needs to deal with the complicated

levels of structure of eukaryotic

chromatin

� Presence of different polymerases whichrequires transcription factors to bind to a

promoter and initiate transcription



RNA POLYMERASE I

� 18S (small), 28S, 5.8S and 5S (large)

� 28S, 18S and 5.8S all come from a large 45Spre-mRNA transcript

� Pol I contains 13 sunbunits (600kDa) and

requires at least 2 TF� The nucleolus is the site of ribosomal subunit

assembly

� About 6800 nucleotides will be discarded inthe process

� The processed rRNAs will combine with 5Sfrom other regions of the nucleus and areexported to the cytosol



RNA POLYMERASE III

� Largest and most complex

� Involves 14 subunits (700kDa), requires

at least 3 TF (TFIIIB,C and A)

� Catallyzes production of tRNAs and 5S

rRNA



RNA POLYMERASE II



FACTOR # of

subunits

MW (kDa) FUNCTION

TFIID(TBP+TAF)

TBP -1TAF -12

3815-250

Core promoter recognition; TFIIBrecruitment

Core promoter recognition; + and ±

regulatory functions

TFIIA 3 12,19,35 Stabilization of TBP binding; stabilization of

TAF-DNA intxn

TFIIB 1 35 RNA polII-TFIIF recruitment ; start-site

selection by RNA pol II

TFIIF 2 30,74 Promoter targeting of polII; destabilization of

nonspecific polII-DNA intxn

RNA polII 12 10-220 Catalysis; recruitemnt of TFIIE

TFIIE 2 34,57 TFIIH recruitement, modulation of TFIIH

helicase, ATPase and kinase activities

TFIIH 9 35-89 Promoter melting using helicase activity,



MAJOR PROBLEMS IN

TRANSCRIPTION

� How can transcription factors and

initiation complex bind to DNA in the

presence of nucleosomes?

� How can the actively transcribing

polymerase pass through arrays of

nucleosomes?



PROBABLE ANSWERS

� Presence of hypersensitive sites

y Susceptible to digestion by nucleases

y Appear in the 5¶ flanking regions of the

embryonic genes

y These provide points at which TF and other

trans-acting proteins can gain access to

promoters and enhancers, allowing initiation

and stimulation of transcription



PROBABLE ANSWERS

� Chromatin Remodellingy Chromatin remodelling factors

y SWI/SNF from yeast and NURF from Drosophil a

y

Both require ATP

hydrolysisy Exact mechanism is still unclear

y Histone acetyltransferase and deacetylase

y High acetylation of histone increasestranscriptional activity

y Acetylation of histones in promoter nucleosomescontributes to loosening of chromatin structures inthese regions



TERMINATION

� The eukaryotic pol II continues totranscribe well past the end of the gene

� In doing so, it passes through 1 or more

AATAAA signals� The pre-mRNA carrying this signal as

AAUAAA is cleaved by specialendonuclease

� After cutting at a site 11 to 30 residues 3¶to it, a poly A tail is added by the non-template directed polymerase



POST TRANSCRITIONAL

PROCESSING

� Addition of 5¶ capping

� Splicing

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