1. 基因表达和调控

3. 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

4. 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

5. 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

6. Regulation of Gene Expression
Operons: Fine Control of
Prokaryotic Transcription

7. 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

8. The lac operon repression model
In the absence of lac

9. 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.

10. In the presence of lactose
The true inducer is allolactose

11. Regulation of Gene Expression in Eukaryote

12. 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

13. Levels of gene expression control in eukaryotes
mRNA Transcription
mRNA processing
mRNA transport
mRNA translation
mRNA degradation
protein processing
protein degradation

14. Transcriptional Control Short Term Regulation
Transcriptional control regulates whether a gene is transcribed and the rate at which transcripts are produced

15. Transcriptional Control
Positive (negative) regulatory element (TATA, etc.)
Positive (negative) regulatory protein

16. Transcriptional Control
Protein coding gene has
1. Core promoter elements: RNAPII, TFs
2. Regulatory elements: regulatory Proteins
3. Enhancers: regulatory Proteins

17. Core promoter elements DNA sequences for RNAP II and TFIIs

18. A model of typical gene & regulatory elements

19. 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:

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

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

22. 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.

23. DNA binding domain: Zn finger
Zn binding involves two Cys and two His a.a.
the finger is a coiled coil
binds to the major groove of DNA double helix

24. 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

25. 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.

26. 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

27. 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

28. 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

29. 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

30. DNA Methylation and T/C Regulation

31. Cytosine (in DNA) methylation
There are many types of DNA methylase (DNMT)

32. 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)

33. 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?

34. 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

35. Epigenetics Chromosome Remodelling Histone Acetylation and Methylation
DNA Methylation

36. RNA Processing Control

37. 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

38. 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

39. Processing control model
A) control by polyA choice
Processing control model
B) control by splices
site choice

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

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

42. RNA Transport Control
Perhaps there are up to 50% protein coding primary RNAs never leave nucleus, degraded.

43. 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

44. mRNA Translation Control

45. Poly(A) tail can promote translation
In general, stored inactive mRNA has shorter PolyA tails (15-90 As) than active mRNAs ( As)
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 ( As),
2. The mature stored mRNAs has short tail (40-60 As)
3. Actively translated mRNAs have As

46. 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

47. mRNA degradation control

48. 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

49. mRNA degradation control
mRNA Tissue or Cell Regulatory Signal Half-Life of mRNA
Vitellogenin Liver (frog) Estrogen h (16h)
Vitellogenin Liver (hen) Estrogen h (<3h)
Lipoprotein Liver (frog) Estrogen h (<3h)

50. 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

51. Protein degradation control

52. 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

53. 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

54. Ubiquitin Tagging
A protein tagged for destruction often requires several molecules of Ubiquitin.
E3 is the reader of the AA

55. The simplest proteasome is found in the archaea
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 Apr 28;268(5210):533-9.

56. 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.

57. RNA interference (RNAi)

58. 中心法则

59. New roles for RNA small RNA RNA ncRNA mRNA rRNA tRNA snRNA snoRNA
Non-coding RNA. Transcribed RNA with a structural,
functional or catalytic role
rRNA
Ribosomal RNA
Participate in
protein synthesis
tRNA
Transfer RNA
Interface between
mRNA &
amino acids
snRNA
Small nuclear RNA
Incl. RNA that
form part of the
spliceosome
snoRNA
Small nucleolar RNA
Found in nucleolus,
involved in modification
of rRNA
small RNA
Other
Including large RNA
with roles in
chromotin structure and
imprinting
siRNA
Small interfering RNA
Active molecules in
RNA interference
miRNA
Micro RNA
Small RNA involved
regulation of expression

60. RNA干扰获得诺贝尔生理学或医学奖
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)。
安德鲁•菲尔(AndrewZ.Fire) 克雷格•梅洛(Craig C.Mello)

61. 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 Feb 19;391(6669):

62. Mex-3 mRNA detection in embryos by in situ hybridization
RAN干扰的发现实验
Double-stranded RNA
control: not stained wt
sense
antisense
inject
wt + antisense RNA
wt + ds RNA
Mex-3 mRNA detection in embryos by in situ hybridization
C. elegans
Nature :

63. RNA干扰的细胞生物学途径 Dicer： A specific RNAase III enzyme
RISC （RNA-induced silencing complex）
RISC*：active forms of RISC complex

64. Model for RNAi
siRNA

65. Dicer contains two RNAse III domains
long dsRNA
siRNAs

66. siRNAs have a defined structure
19 nt duplex
2 nt 3’ overhangs
Guide RNAs
Small ~ 22 nucleotide RNAs associate with RISC

67. RISC –nuclease complex
(RNA-induced silencing complex)
Effector 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

68. RISC –nuclease complex
Latent RISC
Precursor RISC ~ 250K
associate with ds siRNAs
+ATP
Active RISC
Active RISC ~100K
(siRNA unwinding)
associate with ss siRNAs
– destroys target mRNAs
Hannon Review

69. Mechanism of RNAi: Gene Silencing directed by ~22nt RNAs
Amplification of signal:
siRNA may work as primers on the mRNA
Amplification by RNA dependent RNA polymerase

70. 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., ]
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., ]
Non coding, 21nt RNA
Regulates lin-14 in same way as lin-4

71. 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

72. 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.

73. Pathway of siRNAs and miRNAs

74. 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
Imperfect match
 Block translation
Near-perfect match
 Degrade mRNA

75. Endogenous vs exogenous

76. 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.

77. 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).

78. 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