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Gene expression

-overall process by which the information encodedin a gene is converted into an observable phenotype

(most commonly production of a protein).

Why- regulate gene expression? To adjust to sudden changes To conserve energy

To save resources

Regulation of Transcription Initiation

Gene control- All of the mechanisms involved in regulating gene

expression. Most common is regulation of transcription, although

mechanisms influencing the processing, stabilization, and translation

of mRNAs help control expression of some genes.
http://www.ncbi.nlm.nih.gov/books/bv.fcgi?rid=mcb.glossdiv.7888http://www.ncbi.nlm.nih.gov/books/bv.fcgi?rid=mcb.glossdiv.7880http://www.ncbi.nlm.nih.gov/books/bv.fcgi?rid=mcb.glossdiv.7880http://www.ncbi.nlm.nih.gov/books/bv.fcgi?rid=mcb.glossdiv.7880http://www.ncbi.nlm.nih.gov/books/bv.fcgi?rid=mcb.glossdiv.7880http://www.ncbi.nlm.nih.gov/books/bv.fcgi?rid=mcb.glossdiv.7888

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TYPE OF RNA FUNCTION

mRNAs messenger RNAs, code for proteins

rRNAs ribosomal RNAs, form the basic structure of

the ribosome and catalyze protein synthesis

tRNAs transfer RNAs, central to protein synthesis as

adaptors between mRNA and amino acids

snRNAs small nuclear RNAs, function in a variety of

nuclear processes, including the splicing of

pre-mRNA

snoRNAs small nucleolar RNAs, used to process and

chemically modify rRNAs

Other noncoding RNAs function in diverse cellular processes,

including telomere synthesis, X-chromosome

inactivation, and the transport of proteins into

the ER

Table 6-1. Principal Types of RNAs Produced in Cells

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Regulation of gene expression allows cells to adapt to changes in their

environments and is responsible for the distinct activities of the multiple

differentiated cell types that make up complex plants and animals.

Control of transcription initiationthe first stepis the most important

mechanism for determining whether or not most genes are expressed and how

much of the encoded mRNAs, and consequently proteins, are produced.

Transcription

- a process in which a DNA strand provides the informationfor the synthesis of an RNA strand.

-enzymes responsible for transcription in both prokaryoticand eukaryotic cells are called DNA-dependent RNA polymerases

or simply RNA polymerases.

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Transcription in ProkaryotesRNA polymerase- An enzyme that catalyzes the synthesis of RNA.

-the complete enzyme

consists of five subunits: two

, one , one , and one .

-catalyzes the growth of RNAchains always in the 5 to 3

direction (similar to that of DNA

pol)

-however, RNA polymerase does

not require a preformed primerto initiate the synthesis of RNA.

-subunit is required to identify

the correct sites for transcription

initiation

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Promoter -a DNA sequence to which RNA polymerase binds to initiate

transcription.Figure 6.2. Sequences ofE.coli

promotersE. colipromoters

are characterized by two setsof sequences located 10 and 35

base pairs upstream of the

transcription start site (+1). The

consensus sequences shown

correspond to the bases most

frequently found in differentpromoters.

*role ofis to direct the polymerase to promoters by binding specifically

to both the -35 and -10 sequences, leading to the initiation of

transcription at the beginning of a gene

Pribnow box
http://www.ncbi.nlm.nih.gov/books/bv.fcgi?rid=cooper.glossary.2886http://www.ncbi.nlm.nih.gov/books/bv.fcgi?rid=cooper.glossary.2886

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Figure 6.4. Transcription by

E. coliRNA polymerase

a. the polymerase initially

binds nonspecifically to

DNA and migrates along the

molecule until the subunit

binds to the -35 and -10

promoter elements,

forming a closed-promoter

complex.

b. the polymerase then

unwinds (15 bases) DNA

around the initiation site

c. transcription is initiated

by the polymerization of 2

free NTPs.d. subunit then

dissociates from the core

polymerase, which migrates

along the DNA and

elongates the growing RNA

chain.
http://www.ncbi.nlm.nih.gov/books/bv.fcgi?rid=cooper.glossary.2886http://www.ncbi.nlm.nih.gov/books/bv.fcgi?rid=cooper.glossary.2886

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RNA Chain Termination - Termination of RNA chain synthesis appears to be

brought about by two types of mechanisms.

In the first type the termination signal appears to be recognized by DNA

itself. RNA polymerase reads an extended poly (A) sequence on DNA. This

results in an RNA transcript with a terminal poly(U) sequence

The second type of termination signal involves an additional protein called

the rho (P) factor.Rhofactor is an essential transcription protein in

prokaryotes. In Escherichia coli, it is a ~275 kD hexamer of identical subunits.

Each subunit has an RNA-binding domain and an ATP-hydrolysis domain.

Rho is a member of the family of ATP-dependent hexameric helicases that

function by passing nucleic acids through the hole in the middle of the

hexamer. Rho functions as an ancillary factor for RNA polymerase. Rho-

dependent terminators account for about half ofE. coliterminators. The

(rho) factor probably binds to RNA polymerase. It is however, not certain

whether it also, or exclusively, binds to DNA.
http://en.wikipedia.org/wiki/Escherichia_colihttp://en.wikipedia.org/wiki/Subunitshttp://en.wikipedia.org/wiki/RNAhttp://en.wikipedia.org/wiki/Domain_(biology)http://en.wikipedia.org/wiki/Adenosine_triphosphatehttp://en.wikipedia.org/wiki/Helicasehttp://en.wikipedia.org/wiki/Nucleic_acidhttp://en.wikipedia.org/w/index.php?title=Ancillary_factor&action=edit&redlink=1http://en.wikipedia.org/wiki/RNA_polymerasehttp://en.wikipedia.org/wiki/Terminator_(genetics)http://en.wikipedia.org/wiki/Terminator_(genetics)http://en.wikipedia.org/wiki/RNA_polymerasehttp://en.wikipedia.org/w/index.php?title=Ancillary_factor&action=edit&redlink=1http://en.wikipedia.org/wiki/Nucleic_acidhttp://en.wikipedia.org/wiki/Helicasehttp://en.wikipedia.org/wiki/Adenosine_triphosphatehttp://en.wikipedia.org/wiki/Domain_(biology)http://en.wikipedia.org/wiki/RNAhttp://en.wikipedia.org/wiki/Subunitshttp://en.wikipedia.org/wiki/Escherichia_coli

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1. Intrinsic termination (also called Rho-independent termination) is a

mechanism in both eukaryotes and prokaryotes that causes mRNAtranscription

to be stopped. In this mechanism, the mRNA contains a sequence that can base

pair with itself to form a stem-loop structure 7-20 base pairs in length that is also

rich in Cytosine-Guanine base pairs.

Figure 6.5. Transcription

termination The termination

of transcription is signaled bya GC-rich inverted repeat

followed by four A residues.

The inverted repeat forms a

stable stem-loop structure in

the RNA, causing the RNA to

dissociate from the DNA

template.
http://en.wikipedia.org/wiki/Eukaryoteshttp://en.wikipedia.org/wiki/Prokaryoteshttp://en.wikipedia.org/wiki/MRNAhttp://en.wikipedia.org/wiki/Transcription_(genetics)http://en.wikipedia.org/wiki/Stem-loophttp://en.wikipedia.org/wiki/Cytosinehttp://en.wikipedia.org/wiki/Guaninehttp://en.wikipedia.org/wiki/Guaninehttp://en.wikipedia.org/wiki/Cytosinehttp://en.wikipedia.org/wiki/Stem-loophttp://en.wikipedia.org/wiki/Stem-loophttp://en.wikipedia.org/wiki/Stem-loophttp://en.wikipedia.org/wiki/Transcription_(genetics)http://en.wikipedia.org/wiki/MRNAhttp://en.wikipedia.org/wiki/Prokaryoteshttp://en.wikipedia.org/wiki/Eukaryotes

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2. Rho Dependent Polarity

In Prokaryotes transcription and translation are coupled -- that is, ribosomes

begin translation while the mRNA is being produced.

Coupled transcription and translation. During transcription RNA polymerase (RNAP)

separates the two DNA strands and forms a short region of base pairing between the coding

strand of DNA and the newly synthesized RNA. As the RNA elongates it is displaced as the

single-stranded DNA rehybridizes to form double-stranded DNA. Ribosomes rapidly bind to

the resulting single-stranded RNA and begin protein synthesis.

Rho dependent polarity. The process of Rho

dependent polarity is shown stepwise .Rho

dependent polarity is usually initiated by

premature transcription termination.

When ribosomes encounter a stop (aka

"nonsense") codon, they fall off of the RNA.

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Rho factor then interacts with RNAP, causing it to

fall off of the DNA which terminates

transcription. Thus, when translation termination

occurs within a gene it can cause transcriptional

termination, preventing expression of

downstream genes. This process is calledtranslational olarit .

Rho dependent polarity is clearly more

complex than the model shown above.

There is biochemical and genetic evidencethat Rho factor interacts with other

transcription termination factors (for

example, Nus) and the alpha subunit of

RNAP.

Rho factor cannot bind to RNA coated with

ribosomes, but Rho factor binds to specific

regions in naked RNA.

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Concept 1: Gene Regulation in Bacteria

Bacteria adapt to changes in their surroundings by usingregulatory proteins to turn groups of genes on and off in

response to various environmental signals.

The DNA ofEscherichia coliis sufficient to encode about 4000proteins, but only a fraction of these are made at any one time. E.coliregulates the expression of many of its genes according to the

food sources that are available to it.

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Concept 2: The Lactose Operon (Jacob-Monod model)-An operon is a cluster of bacterial genes

along with an adjacent promoter that controls the

transcription of those genes.

When the genes in an operon are transcribed, a singlemRNA is produced for all the genes in that operon. ThismRNA is said to be polycistronic because it carries theinformation for more than one type of protein.
http://www.phschool.com/science/biology_place/biocoach/lacoperon/genereg.html

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An operon

Promoter (DNA) where RNA polymerase binds Operator (DNA) lies between promoter and

structural genes (regulatory

sequence of DNA that controls

transcription of an operon)

Repressor (protein) binds to operator to block

transcription

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The lacoperon of E.colian inducible systemincludes 3 genes:

1. lacZ

encodes -galactosidase (catalyzes the hydrolysisof lactose to glucose and galactose)

2. lacY encodes lactose permease (carrier protein in the

bacterial plasma membrane that moves the sugar into

the cells)3. lacA-encodes thiogalactoside transacetylase (transfers

acetyl group from acetyl-CoA to -galactosides)

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Figure 10-2. Jacob and Monod

model of transcriptional

regulation of the lacoperon by

lacrepressor.

1. When lac repressor binds toa DNA sequence called the

operator (O), which lies just

upstream of the lacZgene,

transcription of the operon

by RNA polymerase is

blocked.

2. 2. Binding of lactose to the

repressor causes a

conformational change in the

repressor, so that it no longer

binds to the operator. RNA

polymerase then is free to

bind to the promoter (P) and

initiate transcription of the

lac genes;

3. the resulting polycistronic

mRNA is translated into theencoded proteins.

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Concept 3: The lacOperator

The operator is a short region of DNA thatlies partially within the promoter and that

interacts with a regulatory protein that controlsthe transcription of the operon.

*Here's an analogy. A promoter is like a doorknob, in that thepromoters of many operons are similar. An operator is like thekeyhole in a doorknob,in that each door is locked by only aspecific key, which in this analogy is a specific regulatory protein.

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Concept 4: The lacRegulatory Gene

The regulatory gene lacIproduces an mRNA that produces a Lac

repressor protein, which can bind to the operator of the lacoperon.

In some texts, the lacI regulatory gene is called the lacI regulator gene. Regulatory genes

are not necessarily close to the operons they affect.

The general term for the product of a regulatory gene is a regulatory protein. The Lac

regulatory protein is called a repressor because it keeps RNA polymerase from

transcribing the structural genes. Thus the Lac repressor inhibits transcription of the lac

operon.

C 5 Th L R P i

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Concept 5: The Lac Repressor Protein

In the absence of lactose, the Lac repressor bindsto the operator and keeps RNA polymerase fromtranscribing the lacgenes.

It would be energetically wasteful for E. coliif the lacgenes wereexpressed when lactose was not present.

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Concept 6: The Effect of Lactose on the lacOperonWhen lactose is present, the lacgenes are expressed

because allolactose binds to the Lac repressor protein andkeeps it from binding to the lacoperator.Allolactose is an isomer of lactose. Small amounts of allolactose areformed when lactose enters E. coli.

Allolactose binds to an allosteric site on the repressor protein causing a conformational

change. As a result of this change, the repressor can no longer bind to the operator region

and falls off. RNA polymerase can then bind to the promoter and transcribe the lac genes.

C t 7 Th l I d All l t

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Concept 7: The lacInducer: Allolactose

Allolactose is called an inducer because itturns on, or induces the expression of, the lacgenes.

The presence of lactose (and thus allolactose) determines whether or not the Lac

repressor is bound to the operator.

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Allolactose binds to an allosteric site on the repressor protein causing a

conformational change. As a result of this change, the repressor can no longer

bind to the operator region and falls off. RNA polymerase can then bind to the

promoter and transcribe the lac genes.

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Concept 8: Feedback Control of the lacOperonWhen the enzymes encoded by the lacoperon

are produced, they break down lactose and

allolactose, eventually releasing the repressor tostop additional synthesis oflacmRNA.

Messenger RNA breaks down after a relatively short amount of time.

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Concept 9: EnergySource Preferences ofE. coli

Whenever glucose is present, E. colimetabolizes it before using alternative energy

sources such as lactose, arabinose, galactose, andmaltose.

Glucose is the preferred and most frequently available energy source for E. coli. The enzymes

to metabolize glucose are made constantly by E. coli.

When both glucose and lactose are available, the genes for lactose metabolism are

transcribed at low levels.

Only when the supply of glucose has been exhausted does does RNA polymerase start to

transcribe the lac genes efficiently, which allows E. colito metabolize lactose.

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Concept 10: The Effect of Glucose and Lactose on the lacOperon

When both glucose and lactose are present, the genesfor lactose metabolism are transcribed to a small extent.

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Maximal transcription of the lacoperon occurs only when glucoseis absent and lactose is present. The action ofcyclic AMP and acatabolite activator protein produce this effect.

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Concept 11: The Effect of Glucose and Cyclic AMP on the lacOperon

The presence or absence of glucose affects the lacoperon by affecting the concentration of cyclic AMP.

*The concentration of cyclic AMP in E. coliis inversely proportional tothe concentration of glucose: as the concentration of glucosedecreases, the concentration of cyclic AMP increases.

Cyclic AMP is derived from ATP.

Concept 12: The Effect of Lactose in the Absence of Glucose

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Concept 12: The Effect of Lactose in the Absence of Glucoseon the lacOperon

In the presence of lactose and absence of glucose,cyclic AMP (cAMP) joins with a catabolite activator proteinthat binds to the lacpromoter and facilitates the

transcription of the lacoperon.In some texts, the catabolite activator protein (CAP) is called thecAMP-receptor protein.

When the

concentration ofglucose is low, cAMP

accumulates in the

cell. The binding of

cAMP and the

catabolite activator

protein to the lacpromoter increases

transcription by

enhancing the

binding of RNA

polymerase to the

lac promoter.

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The lac operon summary

In the absence of inducer, the operon is turned off

Control is exerted by a regulatory protein- the repressor-

that turns the operon off.

Regulatory genes produce proteins whose sole function is to

regulate the expression of other genes.

Certain other DNA sequences (operators and promoters) do notcode for proteins, but are binding sites for regulatory or

other proteins.

Adding inducers turns the operon on.

Operator-repressor control that induces transcription.

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The trp operon ofE. colia repressible system

In repressible systems, the repressor protein cannot shut off itsoperon unless it first binds to a corepressor, which may be

either the metabolic end product itself (tryptophan in this

case) or an analog of it.

Operator-repressor control that represses transcription

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1. Regulatory gene r produces an inactive

repressor, which cannot bind to the

operator

mRNA

Inactive

repressor

PTrp o e d c b a

RNA polymeraseTranscription

proceeds

DNA

mRNA transcript

Translation

e d c b a

Tryptophan absent

DNA

r

Enzymes of the tryptophan

Synthesis pathway

2. RNA polymerase transcribes the

structural genes. Translation

makes the enzymes of the

tryptophan synthesis pathway.

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mRNA

Inactive

repressor

PTrp o e d c b a

DNA

Tryptophan present

DNA

rCorepressor

(tryptophan)

Active repressor

1. Tryptophan binds the

repressor

2. which then binds

to the operator.3. Trp blocks RNA pol from

binding and transcribing thestructural genes, preventing

synthesis of trp pathway

enzymes

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Inducible vs. Repressible systems

in inducible systems, the substrate of a metabolic

pathway (the inducer) interacts with a regulatory protein(the repressor) to render itincapable of binding to the

operator, thus allowing transcription

in repressible systems, the product of a metabolicpathway (the corepressor) interacts with the regulatory

protein to make it capable of binding to the operator,

thus blocking the transcription

In both kinds of systems, the regulatory molecule

functions by binding to the operator.

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Examples ofNegative control of transcription:

a. inducible lac system

b. repressible trp systemThe two operator-repressor systems because the

regulatory molecule (the repressor) in each case prevents

transcription.

Example ofPositive control of transcription:

a. promoter-catabolite repression system because the regulatory molecule (the CRP-cAMP complex)

enhances transcription.

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Transcription in Eukaryotes

Transcription Initiation in Eucaryotes Requires Many Proteins

-eucaryotic nuclei have three RNA pol (bacteria only one):

a. RNA polymerase I

b. RNA polymerase II

c. and RNA polymerase III

* structurally similar to one another but they transcribe different types

of genes

TYPE OF POLYMERASE GENES TRANSCRIBED

RNA polymerase I 5.8S, 18S, and 28S rRNA genes

RNA polymerase II all protein-coding genes, plus

snoRNAgenes and some snRNA genes

RNA polymerase III tRNA genes, 5S rRNA genes, some snRNA

genes and genes for other small RNAs

Table 6-2. The Three RNA Polymerases in Eucaryotic Cells

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Transcription factor (TF) - General term for anyprotein, other than RNA polymerase, required to

initiate or regulate transcription in eukaryotic

cells. General factors, required for transcription

of all genes, participate in formation of the

transcription-initiation complex near the start

site. Specific factors stimulate (or repress)

transcription of particular genes by binding totheir regulatory sequences.

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Several important differences in the way in which the bacterial and

eucaryotic enzymes function:

1. While bacterial RNA polymerase (with factor as one of its subunits) is able

to initiate transcription on a DNA template in vitro without the help ofadditional proteins, eucaryotic RNA polymerases cannot. They require the

help of a large set of proteins called general transcription factors, which must

assemble at the promoter with the polymerase before the polymerase can

begin transcription.

2. Eucaryotic transcription initiation must deal with the packing of DNA intonucleosomes and higher order forms of chromatin structure, features absent

from bacterial chromosomes.

RNA Polymerase II Requires General Transcription Factors

General transcription factor -Any of the proteins whose assembly around theTATA box is required for the initiation of transcription of most eucaryotic

genes.

TATA box -Consensus sequence in the promoter region of many eucaryotic

genes that binds a general transcription factor and hence specifies the

position at which transcription is initiated.

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TATA-binding protein (TBP) -A basal transcription factor that binds directly to the TATA

box.

TFIID (Transcription factorIID) is itself composed of multiple subunits, including

the TATA-binding protein (TBP), which binds specifically to the TATAA consensus

sequence, and 10-12 other polypeptides, called TBP-associated factors (TAFs)

Figure 6.14. RNA polymerase

II holoenzyme The

holoenzyme consists of a

preformed complex of RNApolymerase II, the general

transcription factors TFIIB,

TFIIE, TFIIF, and TFIIH, and

several other proteins that

activate transcription. Thiscomplex can be recruited

directly to a promoter via

interaction with TFIID (TBP +

TAFs).
http://www.ncbi.nlm.nih.gov/books/bv.fcgi?rid=cooper.glossary.2886http://www.ncbi.nlm.nih.gov/books/bv.fcgi?rid=cooper.glossary.2886http://www.ncbi.nlm.nih.gov/books/bv.fcgi?rid=cooper.glossary.2886http://www.ncbi.nlm.nih.gov/books/bv.fcgi?rid=cooper.glossary.2886

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Figure 6-16. Initiation of transcription of a

eucaryotic gene by RNA polymerase II. To

begin transcription, RNA polymerase

requires a number of general transcription

factors (called TFIIA, TFIIB, and so on). (A)

The promoter contains a DNA sequencecalled the TATA box, which is located 25

nucleotides away from the site at which

transcription is initiated. (B) The TATA box is

recognized and bound by transcription

factor TFIID, which then enables the

adjacent binding of TFIIB (C). For simplicity

the DNA distortion produced by the binding

of TFIID (see Figure 6-18) is not shown. (D)

The rest of the general transcription factors,

as well as the RNA polymerase itself,

assemble at the promoter. (E) TFIIH then

uses ATP to pry apart the DNA double helix

at the transcription start point, allowingtranscription to begin. TFIIH also

phosphorylates RNA polymerase II,

changing its conformation so that the

polymerase is released from the general

factors and can begin the elongation phase

of transcription.
http://www.ncbi.nlm.nih.gov/books/bv.fcgi?rid=mboc4.figgrp.1002http://www.ncbi.nlm.nih.gov/books/bv.fcgi?rid=mboc4.figgrp.1002http://www.ncbi.nlm.nih.gov/books/bv.fcgi?rid=mboc4.figgrp.1002http://www.ncbi.nlm.nih.gov/books/bv.fcgi?rid=mboc4.figgrp.1002

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TFIIH is a multisubunit factor that appears to play at least twoimportant roles:

a. First, two subunits of TFIIH are helicases, which may

unwind DNA around the initiation site. (These subunits of TFIIH are

also required for nucleotide excision repair).

b. Another subunit of TFIIH is a protein kinase that

phosphorylates repeated sequences present in the C-terminal

domain of the largest subunit of RNA polymerase II.

Phosphorylation of these sequences is thought to release the

polymerase from its association with the initiation complex,allowing it to proceed along the template as it elongates the

growing RNA chain

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Figure 6.12. Formation of a

polymerase II transcription

complex Many polymerase IIpromoters have a TATA box

(consensus sequence TATAA) 25

to 30 nucleotides upstream of

the transcription start site. This

sequence is recognized bytranscription factor TFIID, which

consists of the TATA-binding

protein (TBP) and TBP-

associated factors (TAFs).

TFIIB(B) then binds to TBP,

followed by binding of the

polymerase in association with

TFIIF(F). Finally, TFIIE(E) and

TFIIH(H) associate with the

complex.

Figure 10-6. DNase I footprinting, a

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common technique for identifying

protein-binding sites in DNA.

a. A DNA fragment is labeled at one

end with 32P (red dot) as in the

Maxam-Gilbert sequencing method.b. Portions of the sample then are

digested with DNase I in the

presence and absence of a protein

that binds to a specific sequence in

the fragment.

(Bottom) Diagram of hypothetical

autoradiogram of the gel for the minus

protein sample above reveals bands

corresponding to all possible

fragments produced by DNase I

cleavage (lane). In the sample

digested in the presence of a DNA-

binding protein, two bands are missing

(+lane); these correspond to the DNA

region protected from digestion by

bound protein and are referred to as

the footprint of that protein.

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Polymerase II Also Requires Activator, Mediator, and Chromatin-

modifying Proteins

DNA in eucaryotic cells is packaged into nucleosomes, which are further arranged

in higher-order chromatin structures. As a result, transcription initiation in a

eucaryotic cell is more complex and requires more proteins

1. gene regulatory proteins known as transcriptional activatorsbind to

specific sequences in DNA and help to attract RNA polymerase II to the start point

of transcription (This attraction is needed to help the RNA polymerase and the

general transcription factors in overcoming the difficulty of binding to DNA that ispackaged in chromatin).

2. eucaryotic transcription initiation in vivo requires the presence of a

protein complex known as the mediator, which allows the activator proteins to

communicate properly with the polymerase II and with the general transcription

factors.3. transcription initiation in the cell often requires the local recruitment of

chromatin-modifying enzymes, including chromatin remodeling complexes and

histone acetylases (both types of enzymes can allow greater accessibility to the

DNA present in chromatin, and so, they facilitate the assembly of the transcription

initiation machinery onto DNA).

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A typical eucaryotic gene

has many activator

proteins, which together

determine its rate and

pattern of transcription.Sometimes acting from a

distance of several

thousand nucleotide pairs

(indicated by the dashed

DNA molecule), these gene

regulatory proteins helpRNA polymerase, the

general factors, and the

mediator all to assemble at

the promoter.

*activators attract ATP-

dependent chromatin-

remodeling complexes and

histone acetylases.

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Protein-coding genes have

exons whose sequence encodes the polypeptide;

introns that will be removed from the mRNA before it is

translated

a transcription start site

a promoter

the basal or core promoter located within about 40 bp of

the start sitean "upstream" promoter, which may extend over as many

as 200 bp farther upstream

enhancers

silencers

Adjacent genes (RNA-coding as well as protein-coding) are

often separated by an insulator which helps them avoid cross-

talk between each other's promoters and enhancers (and/or

silencers).

Enhancers
http://users.rcn.com/jkimball.ma.ultranet/BiologyPages/T/Transcription.htmlhttp://users.rcn.com/jkimball.ma.ultranet/BiologyPages/P/Promoter.htmlhttp://users.rcn.com/jkimball.ma.ultranet/BiologyPages/P/Promoter.htmlhttp://users.rcn.com/jkimball.ma.ultranet/BiologyPages/P/Promoter.htmlhttp://users.rcn.com/jkimball.ma.ultranet/BiologyPages/P/Promoter.htmlhttp://users.rcn.com/jkimball.ma.ultranet/BiologyPages/P/Promoter.htmlhttp://users.rcn.com/jkimball.ma.ultranet/BiologyPages/P/Promoter.htmlhttp://users.rcn.com/jkimball.ma.ultranet/BiologyPages/T/Transcription.html

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Enhancers

Some transcription factors ("Enhancer-binding protein") bind to regions of DNA that

are thousands of base pairs away from the gene they control. Binding increases therate of transcription of the gene.

Enhancers can be located upstream, downstream, or even within the gene they

control.

How does the binding of a protein to an enhancer regulate the transcription of agene thousands of base pairs away?

One possibility is that enhancer-binding proteins in addition to their DNA-

binding site, have sites that bind to transcription factors ("TF") assembled at the

promoter of the gene.

This would draw the DNA into a loop (as shown in the figure).

Visual evidence

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Michael R. Botchan (who kindly supplied these

electron micrographs) and his colleagues have

produced visual evidence of this model of

enhancer action. They created an artificial DNA

molecule with

several (4) promoter sites for Sp1 about 300

bases from one end. Sp1 is a zinc-finger

transcription factor that binds to the sequence 5'

GGGCGG 3' found in the promoters of many genes,

especially "housekeeping" genes.

several (5) enhancer sites about 800 bases from

the other end. These are bound by an enhancer-

binding protein designated E2.

1860 base pairs of DNA between the two.

When these DNA molecules were added to a

mixture of Sp1 and E2, the electron microscope

showed that the DNA was drawn into loops with

"tails" of approximately 300 and 800 base pairs.

At the neck of each loop were two distinguishable

globs of material, one representing Sp1 (red), the

other E2 (blue) molecules. (The two micrographs

are identical; the lower one has been labeled toshow the interpretation.)

Artificial DNA molecules lacking

either the promoter sites or the

enhancer sites, or with mutated

versions of them, failed to form

loops when mixed with the two

proteins.
http://users.rcn.com/jkimball.ma.ultranet/BiologyPages/S/SteroidREs.htmlhttp://users.rcn.com/jkimball.ma.ultranet/BiologyPages/S/SteroidREs.htmlhttp://users.rcn.com/jkimball.ma.ultranet/BiologyPages/S/SteroidREs.htmlhttp://users.rcn.com/jkimball.ma.ultranet/BiologyPages/S/SteroidREs.htmlhttp://users.rcn.com/jkimball.ma.ultranet/BiologyPages/S/SteroidREs.htmlhttp://users.rcn.com/jkimball.ma.ultranet/BiologyPages/S/SteroidREs.html

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SilencersSilencers are control regions of DNA that, like enhancers, may be located

thousands of base pairs away from the gene they control. However, when

transcription factors bind to them, expression of the gene they control is

repressed.

InsulatorsA problem:

As you can see above, enhancers can turn on promoters of genes located

thousands of base pairs away. What is to prevent an enhancer frominappropriately binding to and activating the promoter of some other gene in

the same region of the chromosome?

One answer: an insulator.

Insulators are

stretches of DNA (as few as 42 base pairs may do the trick)located between the

enhancer(s) and promoter or

silencer(s) and promoter

ofadjacent genes or clusters of adjacent genes.

Their function is to prevent a gene from being influenced by the activation (or

repression) of its neighbors.
http://users.rcn.com/jkimball.ma.ultranet/BiologyPages/P/Promoter.htmlhttp://users.rcn.com/jkimball.ma.ultranet/BiologyPages/P/Promoter.html

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Example:

The enhancer for the promoter of the gene for the delta chain of the gamma/delta

T-cell receptor for antigen (TCR) is located close to the promoter for the alpha chain

of the alpha/beta TCR (on chromosome 14 in humans). A T cell must choose

between one or the other. There is an insulator between the alpha gene promoter

and the delta gene promoter that ensures that activation of one does not spread

over to the other.

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All insulators discovered so far in vertebrates work only when bound by a protein

designated CTCF ("CCCTC binding factor"; named for a nucleotide sequence

found in all insulators). CTCF has 11 zinc fingers.

Another example: In mammals (mice, humans, pigs), only the allele for insulin-

like growth factor-2 (IGF2) inherited from one's father is active; that inheritedfrom the mother is not a phenomenon called imprinting.

The mechanism: the mother's allele has an insulator between the IGF2 promoter

and enhancer. So does the father's allele, but in his case, the insulator has been

methylated. CTCF can no longer bind to the insulator, and so the enhancer isnow free to turn on the father's IGF2 promoter.

Many of the commercially-important varieties of pigs have been bred to

contain a gene that increases the ratio ofskeletal muscle to fat. This gene has

been sequenced and turns out to be an allele ofIGF2, which contains a single

point mutation in one of its introns. Pigs with this mutation produce higherlevels ofIGF2 mRNA in their skeletal muscles (but not in their liver).

This tells us that:

Mutations need not be in the protein-coding portion of a gene in order to

affect the phenotype.

Mutations in non-coding portions of a gene can affect how that gene isregulated (here, a change in muscle but not in liver).
http://users.rcn.com/jkimball.ma.ultranet/BiologyPages/M/Muscles.htmlhttp://users.rcn.com/jkimball.ma.ultranet/BiologyPages/M/Mutations.htmlhttp://users.rcn.com/jkimball.ma.ultranet/BiologyPages/P/P.htmlhttp://users.rcn.com/jkimball.ma.ultranet/BiologyPages/P/P.htmlhttp://users.rcn.com/jkimball.ma.ultranet/BiologyPages/M/Mutations.htmlhttp://users.rcn.com/jkimball.ma.ultranet/BiologyPages/M/Muscles.html

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Transcription by RNA Polymerases I and III

All three RNA polymerases, however, require additional transcription

factors to associate with appropriate promoter sequences. Furthermore,

although the three different polymerases in eukaryotic cells recognizedistinct types of promoters, a common transcription factorthe TATA-

binding protein (TBP)appears to be required for initiation of transcription

by all three enzymes.

RNA polymerase I is devoted solely to the transcription of ribosomal RNA

genes, which are present in tandem repeats.

Figure 6.15. The

ribosomal RNA gene The

ribosomal DNA (rDNA) istranscribed to yield a large

RNA molecule (45S pre-

rRNA), which is then

cleaved into 28S, 18S, and

5.8S rRNAs.

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Figure 6.16. Initiation

of rDNA transcription

Two transcription

factors, UBF and SL1,

bind cooperatively to

the rDNA promoter

and recruit RNA

polymerase I to form

an initiation complex.

One subunit of SL1 is

the TATA-binding

protein (TBP).

UBF (upstream binding

factor)

SL1 (selectivity factor 1)

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-promoter for ribosomal RNA genes does not contain a TATA box,

TBP does not bind to specific promoter sequences. Instead, the

association of TBP with ribosomal RNA genes is mediated by the

binding of other proteins in the SL1 complex to the promoter, asituation similar to the association of TBP with the Inr sequences

of polymerase II genes that lack TATA boxes.

-genes for tRNAs, 5S rRNA, and some of the small RNAsinvolved in splicing and protein transport are transcribed

by polymerase III.

-these genes are characterized by promoters that lie

within, rather than upstream of, the transcribedsequence

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Figure 6.17. Transcription of

polymerase III genes The

promoters of 5S rRNA and tRNA

genes are downstream of the

transcrip-tion initiation site.

Transcription of the 5S rRNA

gene is initiated by the binding

of TFIIIA, followed by the binding

of TFIIIC, TFIIIB, and the

polymerase. The tRNApromoters do not contain a

binding site for TFIIIA, and TFIIIA

is not required for their

transcription. Instead, TFIIIC

initiates the transcription of

tRNA genes by binding topromoter sequences, followed

by the association of TFIIIB and

polymerase. The TATA-binding

protein (TBP) is a subunit of

TFIIIB.

RNA Processing and Turnover

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RNA Processing and Turnover

Most newly synthesized RNAs must be modified in various ways

to be converted to their functional forms except bacterial

mRNAs .-the primary transcripts of both rRNAs and tRNAs must undergo

a series of processing steps in prokaryotic as well as eukaryotic

cells.

Processing of Ribosomal and Transfer RNAs

basic processing of rRNA and tRNAs in prokaryotic and eukaryotic

cells is similar, as might be expected given the fundamental roles

of these RNAs in protein synthesis.

*eukaryotes have four species of ribosomal RNAs, three of which (the 28S, 18S,

and 5.8S rRNAs) are derived by cleavage of a single long precursor transcript,

called a pre-rRNA

*prokaryotes have three ribosomal RNAs (23S, 16S, and 5S), which are

equivalent to the 28S, 18S, and 5S rRNAs of eukaryotic cells and are also formed

by the processing of a single pre-rRNA transcript.

RNA Th i t i t hi h i l d t f i di id l ib l

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pre-rRNA -The primary transcript, which is cleaved to form individual ribosomal

RNAs (the 28S, 18S, and 5.8S rRNAs of eukaryotic cells).

Figure 6.37. Processing of

ribosomal RNAs Prokaryotic

cells contain three rRNAs (16S,

23S, and 5S), which are formed

by cleavage of a pre-rRNA

transcript. Eukaryotic cells

(e.g., human cells) contain fourrRNAs. One of these (5S rRNA)

is transcribed from a separate

gene; the other three (18S,

28S, and 5.8S) are derived from

a common pre-rRNA. Followingcleavage, the 5.8S rRNA (which

is unique to eukaryotes)

becomes hydrogen-bonded to

28S rRNA.

tRNAs in both bacteria and

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tRNAs in both bacteria and

eukaryotes are synthesized

as longer precursor

molecules (pre-tRNAs),

some of which contain

several individual tRNA

sequences

Figure 6.38. Processing of transfer RNAs

(A) Transfer RNAs are derived from pre-

tRNAs, some of which contain several

individual tRNA molecules. Cleavage at

the 5 end of the tRNA is catalyzed by theRNase P ribozyme; cleavage at the 3 end

is catalyzed by a conventional protein

RNase. A CCA terminus is then added to

the 3 end of many tRNAs in a

posttranscriptional processing step.

Finally, some bases are modified atcharacteristic positions in the tRNA

molecule. In this example, these

modified nucleosides include

dihydrouridine (DHU), methylguanosine

(mG), inosine (I), ribothymidine (T), and

pseudouridine (y). (B) Structure ofmodified bases. Ribothymidine,

dihydrouridine, and pseudouridine are

formed by modification of uridines in

tRNA. Inosine and methylguanosine are

formed by the modification of

guanosines.

pre-tRNA The primary

transcript, which is cleaved

to form transfer RNAs.

In bacteria, some tRNAs are

included in the pre-rRNA

transcripts.

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RNase P - A ribozyme that cleaves the 5 end of pre-tRNAs.

-consists of RNA and protein molecules, both of which are

required for maximal activity.

ribozyme An RNA enzyme.

Note: an unusual aspect of tRNA processing is the extensive

modification of bases in tRNA molecules. Approximately 10% of the

bases in tRNAs are altered to yield a variety of modified nucleotides

at specific positions in tRNA molecules (see Figure 6.38). The

functions of most of these modified bases are unknown, but some

play important roles in protein synthesis by altering the base-pairingproperties of the tRNA molecule

Transcription Elongation in Eucaryotes Is Tightly Coupled To RNA Processing
http://www.ncbi.nlm.nih.gov/books/bv.fcgi?rid=cooper.figgrp.1030http://www.ncbi.nlm.nih.gov/books/bv.fcgi?rid=cooper.figgrp.1030

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Transcription Elongation in Eucaryotes Is Tightly Coupled To RNA Processing

Figure 6-21. Summary of the

steps leading from gene to

protein in eucaryotes and

bacteria. The final level of aprotein in the cell depends on the

efficiency of each step and on the

rates of degradation of the RNA

and protein molecules. (A) In

eucaryotic cells the RNA molecule

produced by transcription alone

(sometimes referred to as the

primary transcript) would contain

both coding (exon) and

noncoding (intron) sequences.

Before it can be translated into

protein, the two ends of the RNA

are modified, the introns are

removed by an enzymatically

catalyzed RNA splicing reaction,

and the resulting mRNA is

transported from the nucleus tothe cytoplasm.

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Figure 6-23. The RNA factory concept

for eucaryotic RNA polymerase II. Not

only does the polymerase transcribe DNA

into RNA, but it also carries pre-mRNA-

processing proteins on its tail, which are

then transferred to the nascent RNA at

the appropriate time. There are many

RNA-processing enzymes, and not all

travel with the polymerase. For RNA

splicing, for example, only a few critical

components are carried on the tail; oncetransferred to an RNA molecule, they

serve as a nucleation site for the

remaining components. The RNA-

processing proteins first bind to the RNA

polymerase tail when it is phosphorylated

late in the process of transcription

initiation). Once RNA polymerase II

finishes transcribing, it is released from

DNA, the phosphates on its tail are

removed by soluble phosphatases, and it

can reinitiate transcription. Only this

dephosphorylated form of RNA

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