基因表达和调控

The most direct control point

• At the level of transcription: Regulate by changing sets of genes’ expression level in response to environment alteration

Efficiency and economics

Why is Regulation necessary

• Not all genes are expressed continuously: the level of gene expression differ according to--cell types, stages of cell cycle

• Organisms live in changing environments

• Regulation allows organisms to grow and

reproduce optimally in different environments

Are all genes’ activity regulated?

• The housekeeping genes (constitutive genes)

genes that are essential for normal cell function and are constitutionally expressed.

• The regulated genes

Their activity is controlled in response to the needs of a cell or organism

Regulation of Gene Expression

Operons: Fine Control of

Prokaryotic Transcription

• Operons: Fine Control of Prokaryotic Transcription

• The genes, the operator, and the promoter constitute an operon

• The genes are adjacent to each other and are transcribed into a polygenic (polycistronic) mRNA

The lac operon repression model

In the absence of lac

The repressor protein• The repressor protein is a tetramer,

• repressor is an allosteric protein,

with two domains:

a DNA binding domain an inducer binding domain

Inducer binding structural change at DNA binding domain

lose its DNA binding ability

transcription repression

relieved.

In the presence of lactose

The true inducer is allolactose

Regulation of Gene Expression in Eukaryote

Differences between Prokary. and Eukary.

• Prokaryotes: unicellular, free living, gene organization--operons,

control--short term T/C

• Eukaryotes: unicellular or multicellular. No operon organization,

control---short term and long term

• goal: to coordinate the generation of new proteins in different cells at different times

Levels of gene expression control

in eukaryotes

mRNA Transcription

mRNA processing

mRNA transport

mRNA translation

mRNA degradation

protein processing

protein degradation

Transcriptional Control Short Term Regulation

• Transcriptional control regulates whether a gene is transcribed and the rate at which transcripts are produced

Transcriptional Control

• Positive (negative) regulatory element (TATA, etc.)

• Positive (negative) regulatory protein

Transcriptional Control

Protein coding gene has

1. Core promoter elements: RNAPII, TFs

2. Regulatory elements: regulatory Proteins

3. Enhancers: regulatory Proteins

Core promoter elementsDNA sequences for RNAP II and TFIIs

A model of typical gene & regulatory

elements

Transcription machinery binding and Chromosome remodeling

Chromosome remodeling Complex

------ATP-dependent nucleosome remodeling (SWI/SNF)

The effects of modification:

a. Promoter accessibility: ------modification loosens up chromotin structure, increase nucleosome mobility.

b. Attracts some specific DNA binding proteins:

Histone and T/C Regulation Transcription machinery binding and Chromosome remodeling

Histone and T/C Regulation Transcription machinery binding and Chromosome remodeling

Transcription Regulatory Proteins

• DNA binding proteins: recognize specific DNA sequence or structure:

most of them have distinctive structural motifs: Zn finger leucine zipper Helix-Turn-Helix. Some have less distinctive structural motifs, therefore,

have less restrictions for their DNA target.• Some regulatory proteins do not bind to DNA

directly.

DNA binding domain: Zn finger

Zn binding involves two Cys and two His a.a.the finger is a coiled coilbinds to the major groove of DNA double helix

DNA binding domain: leucine zipper

Leucine zipper proteins are dimers, the zipper helix is at the C-terminal of the protein

There are leucine at every 7th position of the helix, face to face in the dimer

The N-terminal helices has + charged amino acids: binding major groove

DNA binding domain: HTH/homeodomain

three short helices separated by turns

the helix proximal to C-terminal is needed for DNA binding

the other two helices needed for protein dimer formation

Drosophila: Homeodomain proteins controls the development of drosophila, all are DNA-binding proteins with HTH motif.

What’s different between specific and non-specific DNA bindings?

• Specific DNA binding proteins interact with the specific bases in a given sequence

• Non-specific DNA binding proteins mostly interact with the phosphate backbone instead of the base

Histone and Gene Regulation

• Histone modification Phosphorylation Acetylation Methylation• Histone must be modified to loosen their grip on the

DNA or be displaced from the DNA so that DNA strands can interact with transcription factors or regulatory proteins

• In essence, the histones act as general repressors of transcription

Histone Acetylation and Transcription Regulation

Active chromatin: Hyper-acetylated• Inactive chromatin: Hypo-acetylated• Acetylation of the lysine at the out sphere of

nucleosome: cause conformational change, destabilizes chromatin structure

• Acetylation make nucleosomes becomes more accessible to transcription factors such as bromodomain bearing TFs

• Protein (histone acetyl transferases,HATs; histone deacetylases, HDACs) are involved in this event

Histone Methylation and Transcription Regulation

Histone methylation and demethylation by histone methyltransferases and histone demethylases are also related to transcription regulation

Certain domains (chromo, tudor etc.) can recognize methylated histone

DNA Methylation and T/C Regulation

Cytosine (in DNA) methylation

There are many types of DNA methylase (DNMT)

DNA Methylation and T/C Regulation

• Methylation could be a signal for DNA involved events: replication, transcription, repair et. al.

(mCpG attract methylation sensitive DNA binding proteins (MeCP1), which in turn recruit Histone de-acetylation enzyme (Sin3 complex) histone de-acetylation gene inactivated

• 5mC : Cytosines are methylated after replication• the percentage of 5mC varies from species to species.(3%

for mammalian DNA, little or no in Drosophila and yeast)• The distribution of 5mC is non-random, Most 5mC was

found in the sequence CG ( mCpG island)

Methylation and T/C regulation

• HpaII/MspI RFLP study of genes with different

activity----negatively correlated (30 genes

examined)

Is this relationship a general picture?

Will all methylated C demethylated in active gene ?

Is methylation level change a necessity of T/C

activity or a byproduct of it?

Over or Under methylation may have serious consequences

• Mutation of methylase in mice is fatal

• Fragile X syndrome: FMR-1(fragile X mental

retardation) gene triplet repeat over expansion

(CGG repeat #>200) and abnormal methylation

------T/C silenced • Abnormal methylation and cancer:

promoter of tumor suppressor gene

Epigenetics

• Chromosome Remodelling

• Histone Acetylation and Methylation

• DNA Methylation

RNA Processing Control

RNA Processing Control

Regulates the production of mature-RNA from precursor-RNA:

1. Choice of alternative poly(A) site

to produce different pre-mRNA molecules

2. Choice of alternative splicing site

to produce different functional mRNAs

RNA Processing Control

The product of alternative poly(A) or alternative splicing are proteins that are encoded by the same gene but differ structurally and functionally

Such proteins are called protein isoforms, and their synthesis may be tissue specific

Alternative poly(A) is independent of alternative splicing

Processing control model

A) control by polyA choice

B) control by splices site choice

• CALC consists of five exons and four introns

• This gene is transcribed in certain cells of the thyroid gland and in certain neurons of the brain

• Alternative PolyA occurs with PolyA site next to exon 4, used in thyroid cells, and PolyA site next to exon 5, used in the neuronal cells

Control by PolyA and Splice site choice — human calcitonin gene (CALC)

Alternative polyadenylation and alternative splicing resulting in tissue-specific products of the human calcitonin gene, CALC

RNA Transport Control

• Perhaps there are up to 50% protein coding primary RNAs never leave nucleus, degraded.

RNA Transport Control

• The spliceosome retention model: spliceosome assembly competes with nuclear

export After splicing process, intron is associated with

snRNPs before degradation

• The methylated 5’cap is necessary for mRNA to be exported to the cytoplasm

mRNA Translation Control

Poly(A) tail can promote translationIn general, stored inactive mRNA has shorter PolyA

tails (15-90 As) than active mRNAs (100-300As)• Is the shorter tail synthesized as it is , or being

truncated to what it is? 1. In oocytes of mouse/frog, the pre-mRNAs has long

tails (300-400 As),

2. The mature stored mRNAs has short tail (40-60 As)

3. Actively translated mRNAs have 100-300 As

Same signal for deadenylation and polyadenylation in cytoplasm

Deadenylation :in the 3’UTR of mRNA, upstream of AAUAAA sequence, there is an (AU)-rich element(ARE) as deadenylation signal (UUUUUAU).

Polyadenylation :to activate a stored mRNA in this class, this signal (ARE element) is recognized by a polyadenylation enzyme and add ~150 As

mRNA degradation control

mRNA degradation control

• Both rRNA and tRNA are very stable, but mRNA exhibits a diverse range of stability

• Regulatory signals change mRNA stability

• mRNA secondary structure & ARE sequence also affect mRNA half life

mRNA Tissue or Cell Regulatory Signal Half-Life of mRNA

Vitellogenin Liver (frog) Estrogen 500h (16h)

Vitellogenin Liver (hen) Estrogen 24h (<3h)

Lipoprotein Liver (frog) Estrogen 20-24h (<3h)

mRNA degradation control

Two mRNA decay pathways• Deadenylation -dependent decay pathway poly(A) tail are deadenylated until the tail are too

short to bind PAB (polyA binding protein) Then 5’cap is removed (decaping) (DCP1) 5’-to-3’ exonuclease degradation

• Deadenylation-independent decay pathway Yeast dcp1 mutant is capable to degrade mRNA Decaping without being deadenylated

Protein degradation control

Protein degradation (proteolysis) control

Balance between the synthesis and degradation

constitutive gene: proteins may be short lived

short lived mRNA: its protein can have long life time

receptors & heat-shock proteins: have short half -lives

Protein degradation (proteolysis) control

• In Eukaryotes, protein degradation require cofactor Ubiquitin

• Ubiquitin: ~8 kd, conservative, C-terminal Gly interacts with the -NH2 of Lys of the targeted protein

Ubiquitin Tagging

A protein tagged for destruction often requires several molecules of Ubiquitin.

E3 is the reader of the AA

The simplest proteasome is found in the archaea. It consists of two types of subunits, organized in the form α7–β7–β7–α7, where each septamer forms a ring. The opening of ~20Å restricts the entrance for substrates.

The top view of the archaeal 20S proteasome shows a hollow cylinder consisting of heptameric rings of α subunits (red) and β subunits (blue). Photograph kindly provided by Robert Huber. The side view of the archaeal 20S proteasome

shows the rings of a subunits (red) and b subunits (blue).

Lowe J, Stock D, Jap B, Zwickl P, Baumeister W, Huber R.Crystal structure of the 20S proteasome from the archaeon T. acidophilum at 3.4 A resolution. Science. 1995 Apr 28;268(5210):533-9.

The eukaryotic 20S proteasome consists of two dimeric rings organized in counter-rotation.

The eukaryotic 26S proteasome is formed when the 19S caps associate with the 20S core, binding to one or both ends, to form an elongated structure of ~45 nm in length.The 19S caps are found only in eukaryotic (not archaeal or bacterial) proteasomes.

RNA interference(RNAi)

RNA 干涉—从发现到获诺贝尔奖

DNA RNA µ°°×Öʸ´ ÖÆת¼ ·­ Òë

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RNA¸´ ÖÆ

中心法

则

New roles for RNA

RNA

mRNAncRNA

Non-coding RNA. Transcribed RNA with a structural, functional or catalytic role

rRNARibosomal RNA

Participate in protein synthesis

tRNATransfer RNA

Interface betweenmRNA &

amino acids

snRNASmall nuclear RNA

-Incl. RNA thatform part of the

spliceosome

snoRNASmall nucleolar RNAFound in nucleolus,

involved in modificationof rRNA

small RNAOther

Including large RNAwith roles in

chromotin structure andimprinting

siRNASmall interfering RNAActive molecules in

RNA interferencemiRNA

Micro RNASmall RNA involved

regulation of expression

RNA 干扰获得诺贝尔生理学或医学奖

安德鲁安德鲁••菲尔菲尔 (AndrewZ.Fire)(AndrewZ.Fire) 克雷格克雷格••梅洛梅洛 (Craig (Craig C.Mello)C.Mello)

2006 年 10 月 2 日瑞典皇家科学院诺贝尔奖委员会宣布，将 2006 年度诺贝尔生理学或医学奖授予两名美国科学安德鲁 · 法尔和克雷格 · 梅洛，以表彰他们发现了 RNA 干扰现象。法尔和梅洛将分享一千万瑞典克朗的奖金 (137 万美元、 107 万欧元 ) 。

RNA 干扰的发现• 安德鲁•菲尔 (AndrewZ.Fire) 、克雷格•梅洛

(Craig C.Mello) 1998 年发现了 RNA 干扰和基因沉默现象。其论文发表在 1998 年的 NATRUE 杂志上。

Fire A, Xu S, Montgomery MK, Kostas SA, Driver SE, Mello CC. Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans.Nature. 1998 Feb

19;391(6669):806-811.

Double-stranded RNA

inject

C. elegans

control: not stainedcontrol: not stained

wt + antisense RNAwt + antisense RNA

sense

antisense

Nature 1998 391:806-811

Mex-3 mRNA detection in embryos by in situ hybridization

wtwt

wt + ds RNAwt + ds RNA

RAN 干扰的发现实验

DicerDicer ： ： A specific A specific RNAase III enzymeRNAase III enzyme

RISC*RISC* ：： active active forms of RISCforms of RISC complexcomplex

RISC RISC （（ RNA-induced RNA-induced silencing complexsilencing complex ））

RNARNA 干扰的干扰的细胞生物学细胞生物学途径途径

siRNA

Model for RNAi

Dicer contains two RNAse III domains

siRNAs

long dsRNA

Guide­RNAsGuide­RNAs Small ~ 22 nucleotide RNAs associate with RISCSmall ~ 22 nucleotide RNAs associate with RISC

19 nt duplex

2 nt 3’ overhangs

siRNAs­have­a­defined­structure

RISCRISC ((RRNA-NA-iinduced nduced ssilencing ilencing ccomplex)omplex)

Effector NucleaseEffector Nuclease

- RISC contains siRNA- precurser activated by ATP- find and destroy mRNA of complementary sequence- contains endo- and exonuclease, cleaves the hybrid in the middle imm. followed by degradation- ARO: PAZ domain

RISC –nuclease complex

Latent RISC Precursor RISC ~ 250Kassociate with ds siRNAs

+ATP

Active RISC Active RISC ~100K (siRNA unwinding)associate with ss siRNAs – destroys target mRNAs

Hannon Review

RISC –nuclease complex

• Amplification of signal:– siRNA may work as primers on the mRNA

• Amplification by RNA dependent RNA polymerase

Mechanism­of­RNAi:­Gene­Silencing­directed­by­~22nt­RNAs

The plot thickens… The Discovery of Endogenous Effectors for RNAi

– Discovery of the first naturally occurring small RNA specie , lin-4• Non-coding, 22nt RNA

– Important for larval development

– lin-4 partially complementary to conserved sites in lin-14 3’UTR [Lee et al., 1993]

• lin-4 binds these sites• lin-4 negatively regulates lin-14 translation

– The naturally occurring small RNA designated microRNAs (miRNAs)

– No other miRNAs found for 7 years!

– Second miRNA – let-7 [Reinhart et al., 2000]• Non coding, 21nt RNA• Regulates lin-14 in same way as lin-4

• miRNA Biogenesis

– Transcribed from endogenous gene as pri-miRNA• Primary miRNA: long with multiple hairpins

• Imperfect internal sequence complementarity

– It is processed into 70-nt hairpins by the RNase III family member Drosha to become the pre-miRNA.

– The pre-miRNA is exported to the cytoplasm by Exportin 5.

– It is cleaved by the R2D2/Dicer heterodimer into the mature miRNA.• Symmetric 2nt 3’ overhangs, 5’ phosphate groups

• The miRNA pathway– pri-miRNA

– processed by Drosha to become the pre-miRNA.

– exported to the cytoplasm by Exportin 5.

– cleaved by the R2D2/Dicer heterodimer into the mature miRNA.

– The miRNA is loaded into RISC and guides it to sites on the mRNA that have only partial sequence complementarity to the miRNA, leading to repression of translation.

Pathway of siRNAs and miRNAs

Imperfect match Block translation

Near-perfect match Degrade mRNA

• miRNA vs. siRNA

– Encoded by endogenous genes vs. Mostly exogenous origin.– Hairpin precursors - pre-miRNAs vs. dsRNA precursors– Translational Repression vs. mRNA cleavage – Recognize multiple targets vs. May be target specific

Endogenous vs exogenous

在当前研究中， miRNA 可以说是热点中的热点。 miRNA 广泛存在于真核生物中 , 本身不具有开放阅读框架 (ORF) ，多数 miRNA 具有高度保守

性， miRNA 基因不是随机排列的 , 其中有一些是成簇的 (cluster) ，而且簇生排列的基因常常协同表达，大多数已发现的 miRNA 的表达都具有时序性。这就意味着 miRNA 可能参与空间发育、应激性、细胞周期和基因重组等

过程。

就在 2005 年，《细胞》杂志上发表文章，美国怀特黑德生物医学研究所和马萨诸塞理工学院的科学家发现，人类基因组中大约有三分之一负责蛋白质合成的基因是由 miRNA 控制的。这一新发现表明，ＲＮＡ在细胞机制中所起的作用远超出先前的认识。除此之外，科学家在《自然》杂志上撰文指出，目前已有的研究结果表明， miRNA 参与控制果蝇的细胞死亡与增殖、哺乳动物的造血作用、线虫的神经网络分布、以及植物的叶和花的发育。现在，一个新的小 RNA 在胰岛瘤细胞中被发现： miR-375 是胰岛特有的，其过度表达抑制由葡萄糖诱导的胰岛素分泌。 miR-375 以及其他组织特定的小

RNA 可能是糖尿病药物治疗的候选目标。有关 miRNA 的研究不但对于我们理解细胞活动中复杂而有序的细节从而探索生命的本质有极大帮助，而且会给医疗的发展提供新的思路和方向。可见

，关于 miRNA 的研究将是生命科学研究的前沿和热点 .

• These "riboregulators" have two traits ideally suited for gene relulation:

1. Being so small, they can be rapidly transcribed from their genes.

2. They do not need to be translated into a protein product to act (in contrast, e.g., to transcription factors).

New Frontiers for RNA…

• Small RNAs likely to have bigger impact on gene and protein regulation

• New classes of small RNAs:

– PiRNA• Small single-strand RNA – 26-31nt• Discovered in a wide range of eukaryotic organisms• Interact with Piwi family proteins• Regulate gene silencing, eg. controlling the transcription and translation of transposons and retrotransposons of

genome