ChapterChapter 17 17 Gene Regulation Gene Regulation

in Eukaryotesin Eukaryotes04 级生物学基地班 姜冠男 200431060045

This chapter can be studied with comparison to chapter 16

F O R E W O R DF O R E W O R D

The regulation in eukaryotes is very like that in prokaryote, but is more complex.

The basic principles and steps of regulation are similar, but eukaryotic genes have additional steps and more regulatory binding sites and are controlled by more regulatory proteins. Besides, nucleosomes and their modifiers influence access to genes.

The increasing complexity of regulatory sequencesfrom a simple bacterial gene controlled by a repressor to a human gene controlled by multiple activators and repressors.

O U T L I N EO U T L I N E Conserved Mechanisms of Transcriptional Regulatio

n from Yeast to Mammals Recruitment of Protein Complexes to Genes by Euka

ryotic Activators Signal Integration and Combinatorial Control Transcriptional Repressors Signal Transduction and the Control of Transcriptiona

l Regulators Gene “Silencing” by Modification of Histones and DN

A Eukaryotic Gene Regulation at Steps after Transcripti

on Initiation RNAs in Gene Regulation

11 CONSERVED MECHANISMS CONSERVED MECHANISMS OF TRANSCRIPTIONAL OF TRANSCRIPTIONAL

REGULATIONREGULATION

FROM YEAST TO FROM YEAST TO MAMMALSMAMMALS

Not all details of gene regulation are the same in all eukaryotes, though they have much in common.

A typical yeast gene has less extensive regulatory sequences than its multicellular counterpart.

Repressors work in a variety of ways.

ⅠActivators have separate DNA binding and activating functions.

The yeast activator Gal4 binds as a dimer to a 17bp site on DNA. The activation domain and the binding domain are separated.

Gal4 binds to four sites located 275bp upstream of Gal1, and activates transcription Of Gal1 gene 1000-fold in the presence of galactose.

Domain swap experiment

(a) The DNA-binding domain of Gal4, without that protein’s activation domain, can still bind DNA, but cannot activate transcription.

(b) Attaching the activationDomain of GA4 to the DNA-binding domain of the bacterial protein LexA, creates a hybrid protein that activates transcription of a gene in yeast as long as that gene bears a binding site for LexA.

The Two Hybrid AssayThis assay is used to identify proteins that interact with each other.

Ⅱ Eukaryotic Regulators Use a Range of DNA-Binding Domains, but DNA Recognition Involves the Same Principlesas Found in Bacteria

Several of the regulatory proteins in eukaryotes bind DNA as heterodimers, and in some cases even as monomers. Heterodimers extend the range of DNA-binding specificities available.•Homeodomain Proteins. The homeodomain is a class of helix-turn-helix DNA-binding domain and recognizes DNA in essentially the same way as those bacterial proteins.•Zinc Containing DNA-Binding Domains. There are various different forms of DNA-binding domain that incorporate a zinc atoms: zinc finger and zinc cluster.•Leucine Zipper Motif. This motif combines dimerization and DNA- binding surfaces within a single structural unit.•Helix-Loop-Helix Proteins. AS in the example of the leucine zipper, an extended ɑ helical region from each of two monomers inserts into the major groove of the DNA.

DNA recognition by a homeodomain.

The homeodomain consists of three ɑ helices, of which two form the structure resembling the helix-turn-helix motif. Thus, helix 3 is therecognition helix and is inserted intothe major groove of DNA.

Zinc finger domain

The ɑ helix on the left of the structure is the recognition helix,and is presented to the DNA bythe β sheet on the right. Thezinc is coordinated by the two His residues in the ɑ helix andtwo Cys residues in the βsheets as shown. Thisarrangement stabilizes the structure and is essential for DNA binding.

zipper bound to DNA.

Two large ɑ helices, one from each monomer, form both the dimerization and DNA-binding domain at different sections along their length. Thus, as shown, toward the top the two helices interact to form a coiled-coil that holds the monomers together; further down, the helices separate enough to embrace the DNA, inserting into the major groove on opposite sides of the DNA-helix. Once again, specificity is provided by contacts made between amino acid side chains on the ɑ helices and the edge of base pairs in the major groove.

Helix-loop-helix motif. A long ɑ helix involved in both DNA recognition and , in combination with a second shorter, ɑ helix dimerization.

Ⅲ Activating Regions Are Not Well-Defined Structures

The activating regions are grouped on the basis of amino acids content•Acidic activation domains •Glutamine-rich domains •Proline-rich domains

22 RECRUITMENT OF PROTEIN RECRUITMENT OF PROTEIN COMPLEXES TO GENESCOMPLEXES TO GENES

BY EUKARYOTIC BY EUKARYOTIC ACTIVATORSACTIVATORS

The activator recruits polymerase indirectly in two ways:

ⅠActivators Recruit the Transcriptional Machinery to the Gene

1 . The activator can interact with parts of the transcription machinery other than polymerase, and , by recruiting them, recruit polymerase as well .2 . Activators can recruit nucleosome modifiers that alter chromatin in the vicinity of a gene and thereby help polymerase bind.In many cases, a given activator can work in both ways.

Activation of transcription initiation in eukaryotes by recruitment of the transcription machinery.

A single activator is shown recruiting two possible target complexes: the Mediator; and, through that, RNA polymerase ; and also the Ⅱgeneral transcription factor TFIID. Other general transcription factors are recruited as part of the Mediator\ Pol complex; separately, or Ⅱbind spontaneously in the presence of the recruited components.

Activation of transcription through direct tethering of mediator to DNA

The GAL1 gene is activated, in the absence of its usual activator Gal4, by thefusion of the DNA-binding domain of LexA to a component of the Mediator Complex. Activation depends on LexA DNA-binding sites being inserted upstream of the gene.Other components required for transcription initiationpresumably bind together with Mediatorand Pol Ⅱ.

At most genes, the transcription machinery is not prebound, and appear at the promoter only uponactivation. Thus, no allosteric activation of the prebound polymerase has been evident in eukaryotic regulation.

ⅡActivators also Recruit Nucleosome Modifiers that Help the Transcription Machinery Bind at the Promoter

Nucleosome modifiers come in two types:•Those that add chemical groups to the tails of histone such as histone acetyl transferases (HATs) , which add acetyl groups;•Those that remodel the nucleosomes, such as the ATP-dependent activity of SWI\ SNF.

How do these modifications help activate a gene?

Two basic models to explain how changes in nucleosomes can help the transcriptional machinery bind at the promoter:•Remodeling, and certain modifications, can uncover DNA-binding sites that would otherwise remain inaccessible within the nucleosome.•Creating specific binding sites on nucleosomes for proteins bearing so-called bromodomains.

Local alterations in chromatin structure directed by activatorsActivators, capable of binding to their sites on DNA within a nucleosome are shown bound upstream of a promoter that is inaccessiblewithin chromain.

(a) The activator is shown recruiting a histone acetylase. That enzyme adds acetyl groups to residues withinthe histone tails. This alters the packing of the nucleosomes somewhat, and also creates binding sites for proteins carrying the appropriate recognition domains.

(b)The activator recruits a nucleosome remodeller, which alters the structure of nucleosomes around the promoter, rendering it accessible and capable of binding the transcription machinery.

Ⅲ Action at a Distance: Loops and Insulators

Many eukaryotic activators---particularly in higher eukaryotes---work from a distance. Various models have been proposed to explain how proteins binding in between enhancers and promoters might help activation in the cells of higher eukaryotes.•In Drosophila, the cut gene is activated from an enhancer some 100 kb away. A protein called Chip aids communication between enhancer and gene.•In eukaryotes, the DNA is wrapped in nucleosomes, and the histones within those nucleosomes are subject to various modifications that affect their disposition and compactness.

Specific elements called Insulators control the actions of activators, preventing the activating of the non-specific genes.

Insulators block activation by enhancers.

a) A promoter activated by activators bound to an enhancer.b) An insulator is placed between the enhancer and the promoter. When bound by appropriate insulator- binding proteins, activation of the promoter by the enhancer is blocked, despite activators binding to the enhancer.c) The activator can activate another promoter nearby.s) The original promoter can be activated by another enhancer placed downstream.

Transcriptional silencingSilencing is a specialized form of repression that can spread along chromatin, switching off multiple genes without the need for each to bear binding sites for specific repressors. Insulator elements can block this spreading, so insulators protect genes from both indiscriminate activation and repression.

Agene inserted at random into the mammalian genome is often “silenced” because incorporated into a particularly dense form of chromatin called heterochromatin. But if insulators are placed up- and downstream of that gene they protect it from silencing.

Ⅳ Appropriate Regulation of Some Groups of Genes Requires Locus Control Regions

Only in adult bone marrow are the correct regulators all active and present in appropriate concentrations to bind these enhancers. But more than this is required to switch on these genes in the correct order.

A group of regulatory elements collectively called the locus control region, or LCR, is found 30-50 kb upstream of the whole cluster of globin genes. It binds regulatory proteins that cause the chromatin structure around the whole globin gene cluster to “OPEN UP”, allowing access to the array of regulators that control expression of the individual genes in a defined order.

Regulation by LCRs

(a)The human globin genes and the LCR that ensures their ordered expression.(b) The globin genes from mice, which are also regulated by an LCR.(C) The HoxD gene cluster from the mouse controlled by an element called the GCR which likethe LCRs appears to impose ordered expression on the gene cluster.

33 SIGNAL INTERATION SIGNAL INTERATION ANDAND

COMBINATORIAL COMBINATORIAL CONTROLCONTROL

Ⅰ Activators Work Together Synergistically to Integrate Signals

In multicellular organisms signal integration is used extensively. In some cases numerous signals are required to switch a gene on. So at many genes multiple activators must work together to switch the gene on. When multiple activators work together, they do so synergistically. That is , the effect of two activators working together is greater than the sum of each of them working alone.

Three strategies of synergy:•Two activators recruit a single complex•Activators help each other binding cooperativity

Cooperative binding of activators

(a)Cooperative binding through direct interaction between the two proteins.(b) A similar effect is achieved by both proteins interacting with a common third protein.(c) The first protein recruits a nucleosome remodeller whose action reveals a binding site for a second protein.(d) Binding a protein unwinds the DNA from nucleosome a little, r revealing the binding site for another protein.

Ⅱ Signal Integration: the HO Gene Is Controlled by Two Regulators; One Recruits Nucleosome Modifiers and the Other Recruits Mediator.

The HO gene is involved in the budding of yeast. The HO gene is expressed only in mother cells and only at a certain point in the cell cycle. These two conditions are communicated to the gene through two activators: SWI5 and SBF.

Why does expression of the gene depend on both activators?•SBF( which is active only at the correct stage of the cell cycle) cannot bind its sites unaided; their disposotion within chromatin prohibits it. •SWI5( Which acts only in the mother cell) can bind to its sites unaided but cannot, from that distance, activate the HO gene.•SWI5 can recruit nucleosome modifiers. These acts on nucleosomes over the SBBF sites.•Thus, if both activators are present and active, the action of SWI5 enables SBF to bind, and that activator, in turn, recruits the transcriptional machinery and activates expression of the gene.

Control of the HO gene

SWI5 can bind its sites within chromatin unaided, but SBF cannot. Remodellers and histone acetylasesrecruited by SWI5alter nucleosomes over the SBF sites, allowing that activator to bind near the promoter and activate the gene.

Ⅲ Signal Integration: Cooperative Binding of Activators at the Human β-Interferon Gene

The human β-interferon gene is activated in cells upon viral infection. Infection triggers three activators: NFĸB, IRF, and Jun\ ATF. They bindcooperatively to sites within an enhancer, form a structure called enhanceosome.

The human β-interferon enhanceosome

Cooperative binding of the three activators, to gether with the architectural protein HMG-1, activates the β-interferon gene.

Ⅳ Combinatorial Control Lies at the Heart of the Complesity and Diversity of Eukaryotes

There is extensive combinatorial control in eukaryotes. In complex multicellular organisms, combinatorial control involves many more regulators and genes, and repressors as well as activators can be involved.

Combinatorial control

Each of the two gene controlled by multiple signals- four in the case of gene A; three in the case of gene B. Each signal is communicated to a gene by one regulatory protein. Regulatory protein 3 acts at both genes, in combination with different additionalRegulators in the two cases.

Ⅴ Combinatorial Control of the Mating-Type Genes from Saccharomyces cerevisiae

The yeast S. cerevisiae exists in three forms: Two haploed cells of different mating types-a and ɑ-and the diploid formed when and an a and an ɑ cell mate and fuse.

Cells of the two mating types differ because they express different sets of gene: a specific genes and ɑ specific genes.

The a cell and the ɑ cell each encode cell type specific regulators:

•a cells make the regulatory protein a1; • ɑ cells make the proteins ɑ1 and ɑ2.•A fourth regulatory protein called Mcm1, is also involved in regulating the mating-type specific genes and is present in both cell types.

Control of cell-type specific genes in yeast

The three cell types of the yeast S. cerevisiae are defined by the sets of genes they express. Oneubiquitous regulator( Mcm1) and three cell-type specific regulators( a1, ɑ1 and ɑ2) together regulate three classes of target genes. The MAT locus is the region of the genome which encode the mating type regulator.

4 4 TRANSCRIPTIONAL TRANSCRIPTIONAL

REPRESSORSREPRESSORS

Repressors don’t work by binding to sites that overlap the promoter and thus block binding of polymerase.

Another form of repression different from bacteria, which is the most common in eukaryotes.It works as follows:As with activators, repressors can recruit nucleosome modifiers, but in this case the enzymes have the opposite effects to those recruited by activators---they compact the chromatin or remove groups recognized by the transcriptional machinery.

Ways in which eukaryotic repressors work

a) By binding to a site on DNA that overlaps the binding site of an activator, a repressor can inhibit binding of the activator to a gene, and thus block activation of that gene.b) A repressor binds to a site on DNA beside an activator and interacts with that activator, occluding its activating region.c) A repressor binds to a site upstream of a gene and, by interacting with thetranscriptional machinery at the promoter in some specific way, inhibits transcription initiation.d) repression by recruiting histone modifiers that alter nucleosomes in ways that inhibit transcription.

Repression of the GAL1 gene in yeast

In the presence of glucose, Mig1 binds a site between the UASG and the GAL1 promoter. By recruiting the Tup1 repressing complex, Mig1 repress expression of GAL1. Repression is a result of deacetylation of local nucleosomes, and also probably by directly contacting and inhibiting the transcription machinery.

5 5 SIGNAL TRANSDUCTION AND SIGNAL TRANSDUCTION AND THE CONTROL OFTHE CONTROL OF

TRANSCRIPTIONAL TRANSCRIPTIONAL REGULATORSREGULATORS

Ⅰ Signals Are Often Communicated to Transcriptional Regulators through Signal Transduction Pathways

The term “signal” refers to the initiating ligand itself---that is , the sugar or protein or others. It can also refer to the “information” as it passes from detection of that ligand to the regulators that directly control the genes---that is, as it passes along a signal transduction pathway.

In a signal transduction pathway, the initiating ligand is typically Detected by a specific cell surface Receptor.

From there the signal is relayed to the relevant transcriptional regulator, often through a cascade of kinases.

Through an allosteric change in the receptor ,the binding of ligand ot the extracellular domain is communicated to the intracellular domain.

Two signal transduction pathways from mammalian cells(a) The STAT pathway (b) The MAP kinase pathway

Ⅱ Signals Control the Activities of Eukaryotic Transcriptional Regulators in a Variety of Ways

Once a signal has been communicated, directly or indirectly, to a transcriptionalregulator, how does it control the activity of that regulator?

In eukaryotes, transcriptional

regulators are not typically

controlled at the level of DNA binding.

Regulators are instead usually controlled

in one of two basic ways:

•Unmasking an Activating Region.

•Transport Into and Out of the Nucleus.

The yeast activator Gal4 is regulated by the Gal80 protein

Activator Gal4 is regulated by masking protein

Ⅲ Activators and Repressors Sometimes Come in Pieces

For example, the DNA binding domain and activating region can be on different polypeptides, which come together on DNA to form the activator.

In addition, the mature of the complex can determine whether the DNA-binding protein activates or represses nearby genes.

66 GENE “SILENCING” BY GENE “SILENCING” BY MODIFICATION OF HISTONESMODIFICATION OF HISTONES

AND DNAAND DNA

Silencing is a position effect---a gene is silenced because of where it is located, not in response to a specific environmental signal. Also, silencing can “spread” over large stretches of DNA ,switching off multiple genes, even ones quite distant from the initiating event.

The most common form of silencing is associated with a dense form of chromatin called heterochromatin. Both activation and repression of transcription often involve modification of nucleosomes to alter the accessibility of a gene to the transcriptional machinery and other regulatory proteins. Transcription can also be silence by methylation of DNA by enzymes called DNA methylases.

Ⅰ Silencing in Yeast Is Mediated by Deacetylation and Methlation of Histones

The telomeres, the silent mating-type locus, and the rDNA genes are all “silent” regions in S. cerevisiae. Methylation of DNA sequence can inhibit binding of proteins, including the transcriptional machinery, and thereby block gene expression.

Consider the telomere as an example.•The final 1-5 kb of each chromosome is found in a folded , dense structure. Genes taken from other chromosomal locations and moved to this region are often silenced, particularly if they are only weakly expresse din their usual location.•Mutations have been isolated in which silencing is relieved ---that is, in which a gene placed at the telomere is expressed at higher levels. These studies implicate three genes encoding regulators of silencing: SIR2,3 and 4. The three proteins encoded by these genes form a complex that associates with silent chromatin, and Sir2 is a histone deacetylase.•The silencing complex is recruited to the telomere by a DNA-binding protein that recognizes the telomere’s repeated sequences.•Histone methyl transferases attach methyl groups to histone tails.•Just as acetylated residues within histones are recognized by proteins bearing bromodomains, methylated residues bind proteins with chromodomains.

Rap1 recruits SIR complex to the telomere. SIR2, a component of that ocmplex, deacetylates nearby nucleosomes, The unacetylated tails themselves then bind Sir3 and SIR4, recruiting more SIR complex, allowing the SIR2 within it to act on nucleosomes further away, and so on. This explains the spreading of the silencing effect produced by deacetylation.

Silencing at the yeast telomere

ⅡHistone Modifications and the Histone Code Hypothesis

According to the histone code edea, different patterns of modifications on histone tails can be “read” to mean different things.The “meaning” would, in part, be the result of the direct effects of these modifications on chromatin density and form.But in addition, the particular pattern of modifications at any given location would recruit specific proteins, the particular set depending on the number, type, and disposition of recognition domains those proteins carry.There are also proteins that phosphorylate serine residues in H3 and H4 tails and proteins that bind thosse modifications.Add to this the observation that many of the proteins that carry modification –recognizing domains are themselves enzymes that modify histones further.

Switching a gene off through DNA methylation and histone midification

Ⅲ DNA Methylation Is Associated with Silenced Genes in Mammalian Cells

Some mammalian genes are kept silent by methylation of nearby DNA sequences. Methylation of DNA can mark sites where heterochromatin subsequently forms.DNA methylation lies at the heart of a phenomenon called imprinting.Two regulatory sequences are critical for the differential expression of the human H19 and Igf2 genes: An enhancer and an insulator.

Imprinting

Two examples of genes controlled by imprinting- the mammalian Igf2 and H19 genes. The H19 genes is expressed from only the matermal chromosome, Igf2 from the paternal chromosome. The methylation state of the insulator element determines whether or not the insulator binding protein( CTCF) can bind and block activation of the H19 gene from the downstream enhancer.

Ⅳ Some States of Gene Expression Are Inherited through Cell Division even when the Initiating Signal Is No Longer Present

Patterns of DNA methylation can be maintained through cell division

77 EUKARYOTIC GENE EUKARYOTIC GENE REGULATION AT STEPSREGULATION AT STEPS

AFTER AFTER TRANSCRIPTION TRANSCRIPTION

INITIATIONINITIATION

Ⅰ Some Activators Control Transcriptional Elongation rather than Initiation

At some genes there are sequences downstream of the promoter that cause pausing or stalling of the polymerase soon after initiation. At those genes, the presence or absence of certain elongation factors greatly influences the level at which the gene is expressed.One example is the HSP70 gene from Drosophila.The HIV virus , which causes AIDS, transcribes its genes from a promoter controlled by P-TEF, which is brought to the stalled polymerase by TAT. Tat recognizes a specific sequence near the start of the HIV RNA and present in the transcript made by the stalled polymerase. Another domain of TAT interacts with P-TEF and recruitsit to the stalled polymerase.

Ⅱ The Regulation of Alternative mRNA Splicing Can Produce Different Protein Products in Different Cell Types

In some cases a given precursor mRNA can be spliced in alternative ways to produce different mRNAs that encode different protein products. The choice of splicing variant produced at a given time or in a given cell type can be regulated.The regulation of alternative splicing works in a manner reminiscent of transcriptional regulation.In other cases, sequences called splicing enhancers are found near splice sites.

The sex of a fly is determined by the ratio of X chromosomes to autosomes. This ratio is initially measured at the level of transcription using two activators SisA and SisB. The genes encoding these regulators are both on the X chromosome, and so , in the early embryo, the prospective female makes twice as much of their products as does the male.These activators bind to sites in the regulatory sequence upstream of the gene Sex-lethal( Sxl). Another regulator that binds to and controls the Sxl gene is a repressor calledDpn( Deadpan); this is encoded by a gene found on one of the autosomes( chromosome2). Thus the ratio of activators to repressor differs in the two sexes, and this makes the difference between the Sxl gene being activated( in females) and repressed( in males).

Early transcriptional regulation of Sxl in male and female flies.

A cascade of alternative splicing events determines the sex of a fly

Ⅲ Expression of the Yeast Transcriptional Activator Gcn4 Is Controlled at the Level of Translation

Gcn 4 is a yeast transcriptional activator that regulates the expression of genes encoding enzymes that direct amino acid biosynthesis. Although it is a transcriptional activator, Gcn4 is itself regulated at the level of translation. The mRNA encoding the Gcn4 protein contains four small open reading frames called uORFs upstream of the coding sequence for Gcn4.The most upstream of these short open-reading frames( Uorf1) is efficiently recognized by ribosomes that scan along the message from the 5’ end. Before initiating translation of any downstream open-reading frame scanning 40s ribisome subunits must bind the translation factor Eif2 complexed with the initiating tRNAs molecule Met-tRNAiMet.

Translational control of Gcn4 in response to amino acid starvation

The open-reading frame encoding the yeast activator Gcn4is preceeded by four other ORFs. The first of these upstream ORFs is translated initially. When amino acids are scarce( starvation conditions), it takes longer for the translational machinery to re-initiate translation, and soit tends to reach the Gcn4-encoding open-reading frame before re-initiating and translates that to give Gcn4 protein, When amino acids are plentiful( nonstarvation conditions) re-initiation takes place at intervening open-reading frames, and the translation machinry then dissociates from the RNA template and Gcn4 is never translated.

High levels of amino acids: the Gcn4 mRNA is not translated

Low levels of amino acids: the Gcn4 mRNA is translated

88 RNAs IN GENERNAs IN GENE

REGULATIONREGULATION

RNAs have a more general and mechanistically distinct role in gene regulation.Short RNAs, generated by the action of enzymes can direct repression of genes with homology to those short RNAs. This repression, called RNA interference( RNAi), can manifest as translational inhibition of the mRNA, destruction of the mRNA or transcriptional silencing of the promoter that directs expression of that mRNA. The role of these RNAs ranges from developmental regulation to the protection against infection by certain viruses.RNAi has also been adapted for use as a powerful experimental technique allowing specific genes to be specific genes to be switched off in any of many organisms.

Ⅰ Double-Stranded RNA Inhibits Expression of Genes Homologous to that RNAThe discovery that simply introducing double-stranded RNA( dsRNA) into a cell can repress genes containing sequences identical to( or very similar to) that dsRNA was remarkable in 1998 when it was reported. In that case, the experiment was done in the worm C. elegans. A similar effect is seen in many other organisms in which it has subsequently been tried. Earlier than this report, however, it had been known that in plants genes could be silenced by copies of homologous genes in the same cell. Those additional transgenes were often found in multiple copies, some Integrated in direct repeat orientation. Also, in plants, it was known that infection by viruses was combated by a mechanism that involved destruction of viral RNA. These two cases were brought together in the following observation: infection of a plant with an RNA virus that carried a copy of an endogenous plant gene led to silencing of that endogenous gene. All these phenomena are now known to be mechanistically linked.

Ⅱ Short Interfering RNAs( siRNA) Are Produced from dsRNA and Direct Machinery that Switches Off Genes in Various Ways

Dicer is an RNAse -like enzyme that recognizes and dⅢigests long dsRNA. The products of this are short double-stranded fragments about 23 nucleotides long. These short RNAs( often called short interfering RNAs, or siRNAs) inhibit expression of a homologous gene in three ways:they trigger destruction of its mRNA; they inhibit translation of its mRNA; or they induce chromatin modifications within the promoter that silence the gene.

That machinery includes a complex called RISC( RNA- induced silencing complex). A RISC complex contains, in addition to the siRNAs themselves, various proteins including members of the Argonaut family, which are believed to interact with the RNA component.There is another feature of RNAi silencing worth noting- its extreme efficiency. Very small amounts of dsRNA are enough to induce complete shutdown of target genes.

Why the effect is so strong?It might involve an RNA-dependent RNA polymerase which is required in many cases of RNAi. The involvement of this enzyme suggests some aspect of the inhibitory “signal” might be amplified as part of the process.

Ⅲ MicroRNAs Control the Expression of some Genes during Development There is another class of maturally occurring RNAs, called microRNAs( miRNAs), that direct repression of genes in plants ans worms.Often these miRNAs are expressed in developmentally regulated patterns.The miRNAs, typically 21 or 22 nts long, arise from larger precursors( about 70-90 nts long) transcribed from non-protein encoding genes.These transcripts contain sequences that form stem loop strutures, which are processed by Dicer. The miRNAs they produce lead to the destruction( typically the case in plants) or translational repression( in worms) of target mRNAs with homology to the miRNA.

Summary1.There are several complexities in the organization and transcription of eukaryotic genes not found in bacteria:• Nucleosomes and their modification.• Many regulators and larger distances.• The elaborate transcriptional machinery.

2.In eukaryotes, activators predominantly work by recruitment, but do not recruit polymerase directly or alone. They recruit the other protein complexes required to initiate transcription of a given gene.

3.Some activators work from sites far from the gene, requiring that the DNA between their binding sites and the promoter loops out.

4.Most commonly, eukaryotic repressors work by recruiting histone modifiers that reduce transcription.

5.Groups of genes can be kept in a “silent” state without the need for specific repressors bund at each individual gene.In some eukaryotic organisms, such as mammals, silent genes are also associated with methylated DNA.

6.There are various steps in gene regulation after transcription initiation can be regulated. These include transcriptional elongation and translation,but most striking is the regulation of splicing.

7.Another form of gene regulation involves small RNA molecules that inhibit expression of homologous genes.

8.The mechanisms by which these RNAs inhibit expression of genes can involve destruction of mRNA, inhibition of translation, and RNA-directed modification of nucleosomes in the promoters of genes.

Important conceptions:

•Promoter •Regulator binding site•Enhancer•Insulator•Reporter gene•Gene silencing

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