1. The Cell Nucleus and the Control of Gene Expression
CHAPTER 12
The Cell Nucleus and the Control of Gene Expression

2. Cells express their genetic information selectively.
Gene expression is controlled by regulatory machinery in the cell nucleus.
Gene regulation
-Prokaryotes
-Eukaryotes
---Genomic control
---Transcriptional control
---Posttranscriptional control

3. 12.2 Control of Gene Expression in Bacteria
Genes
--Constitutive.
(i.e., organic carbon,
nitrogen, phosphorus,
sulfur and metal ions-iron-,
Ribosomal and glycolysis genes).
---Regulated.
(i.e., 02 -metabolic genes-)
---Adaptative.
(i.e., present or absence of
nutrients)

4. Bacterial cells selectively express genes to use the available resources effectively.
The presence of lactose in the medium indices the synthesis of the enzyme β-galactosidase.
The presence of tryptophan in the medium represses the genes that encode enzymes for tryptophan synthesis.

5. The kinetics of β-galactosidase induction
Inducible enzymes
Substrate induction
Catabolic Pathway

6. End-product repression
Anabolic Pathway
Product inhibition
End-product repression
Negative feedback

7. Control of Gene Expression in Bacteria
The Bacterial Operon
An operon is a functional complex of genes containing the information for enzymes of a metabolic pathway. It includes:
Structural genes – code for the enzymes and are translated from a single mRNA (Polycistronic).
Promoter – where the RNA polymerase binds.
Operator – site next to the promoter , where the regulatory protein can bind.
A repressor (~proteins) which binds to a specific DNA sequence to determine whether or not a particular gene is transcribed.
The regulatory gene encodes the repressor protein

8. Organization of a bacterial operon

9. Gene regulation by operons
Inducible operon
Allosteric protein

10. Control of Gene Expression in Bacteria (4)
The lac Operon
It is an inducible operon, which is turned on in the presence of lactose (inducer).
The lac operon contains three structural genes.
Lactose binds to the repressor, changing its conformation and making it unable to bind to the operator.
A repressor protein can bind to the operator and prevent transcription in the absence of lactose.

11. Glucose indirectly inhibits the adenyl cyclase
The cAMP Receptor Protein (CRP) and Its Function
[Glucose]
[cAMP]
Glucose indirectly inhibits the adenyl cyclase
CRP: cAMP receptor protein

12. Control of Gene Expression in Bacteria (5)
The lac Operon (continued)
Positive Control by Cyclic AMP
The lac repressor exerts negative control.
The glucose effect is an example of positive control.
Cyclic AMP (cAMP) acts by binding to a cAMP receptor protein (CRP).
Binding of CRP-cAMP to the lac control region changes the conformation of DNA thus allowing RNA polymerase to transcribe the lac operon.

13. The Tryptophan (trp) Operon of E. coli
Repressible operon

14. Control of Gene Expression in Bacteria (6)
The trp Operon
It is a repressible operon, which is turned off in the presence of tryptophan.
The trp operon repressor is active only when it is bound to a corepressor such as tryptophan.

15. Control of Gene Expression in Bacteria (7)
Riboswitches
A number of bacterial mRNAs can bind to a small metabolite (molecules) , which in turn alters the gene involved in the production of such metabolite.
These mRNAs are called riboswitches because they undergo a conformational change and can suppress gene expression.
Riboswitches allow bacteria to regulate gene expression in response to some metabolites.

16. Riboswitches= small molecules regulating gene expression by interacting mRNA
Prokaryotes/Plants

17. Control of Gene Expression in Eukaryotes (2)
Genes are turned on and off as a result of interaction with regulatory proteins.
Each cell type contains a unique set of proteins.
Regulation of gene expression occurs on three levels:
Transcriptional-level control
Processing-level control
Translational-level control

18. ---Transcriptional control
-Eukaryotes
---Genomic control
---Transcriptional control
Transcriptional-level control
Processing-level control
---Posttranscriptional control

19. -Eukaryotes
---Genomic control
----- gene amplification
(i.e., in same type of cancers--EGFR, IGFR)
-----gene deletion
(i.e., red cells no DNA)
-----gene rearrangements
(i.e., IgG)
-----Chromatin (de)condensation
-----DNA metylation
-----Histone acetylation
---Transcriptional control
---Posttranscriptional control

20. The cell nucleus

21. 12.1 The Nucleus of a Eukaryotic Cell (1)
The contents of the nucleus are enclosed by the nuclear envelope.
A typical non-dividing nucleus includes:
Chromosomes as extended fibers of chromatin.
Nucleoli for rRNA synthesis.
Nucleoplasm as the fluid where solutes are dissolved.
The nuclear matrix, which is the protein-containing fibrillar network.

22. The Nucleus of a Eukaryotic Cell (2)
The Nuclear Envelope
The nuclear envelope is a structure that divides the nucleus from its cytoplasm.
It consists of two membranes separated by a nuclear space.
The two membranes are fuses at sites forming a nuclear pore.
The inner surface of the nuclear envelope is lined by the nuclear lamina.

23. The nuclear envelope

24. The Nucleus of a Eukaryotic Cell (3)
The nuclear lamina
Support the nuclear envelope.
It is composed of lamins.
The integrity of the nuclear lamina is regulated by phosphorylation and dephosphorylation.

25. The nuclear lamina
Hutchinson-Gilford progeria syndrome

26. The Nucleus of a Eukaryotic Cell (4)
The Structure of the Nuclear Pore Complex and its Role in Nucleocytoplasmic Exchange
Proteins and RNA are transported in and out of the nucleus.
Nuclear pores contain the nuclear pore complex (NPC) that appears to fill the pore like a stopper.
NPC is composed of ~30 proteins called nucleoporins.

27. Movement of materials though the nuclear pore

28. A model of the vertebrate NPC
Amino acids

29. The Nucleus of a Eukaryotic Cell (5)
Proteins synthesized in the cytoplasm are targeted for the nucleus by the nuclear localization signal (NLS).
Proteins with an NLS stretch bind to an NLS receptor (importin).
Conformation of the NPC changes as the protein passes through.
RNAs move through the NPCs as RNPs and carry NES (nuclear export signals) to pass through.

30. Importing proteins from the cytoplasm into the nucleus
NES
NLS

31. The Nucleus of a Eukaryotic Cell (6)
Chromosomes and Chromatin
Packaging the Genome
Chromosomes consist of chromatin fibers, composed of DNA and associated proteins.
Each chromosome contains a single, continuous linear DNA molecule.

32. The Nucleus of a Eukaryotic Cell (8)
DNA and histones are organized into repeating subunits called nucleosomes.
Each nucleosome includes a core particle of supercoiled DNA containing H2A, H2B, H3, and H4.
Histone H1 functions as a linker.
The histone core complex consists of two molecules each of H2A, H2B, H3, and H4 forming an octamer.
The protein component of chromosomes include histones, a group of highly conserved proteins.
Histones have a high content of basic amino acids.

33. Nucleosomal organization of chromatin

34. Levels of organization of chromatin

35. The Nucleus of a Eukaryotic Cell (11)
Heterochromatin and Euchromatin
Euchromatin: active DNA
Heterochromatin: inactive DNA
Constitutive heterochromatin remains condensed all the time (centromeres and telomeres). Consists of highly repeated sequences and few active genes (DNA stability).
Facultative heterochromatin is inactivated during certain phases of the organism’s life. X chromosomes inactivation during mammalian embryogenesis.

36. Sensitivity of Active Genes in Chromatin to Digestion with DNase I
-----Chromatin decondensation
Decondensation ~ gene transcription
Hypersensitive sites (DNAase)

37. The Nucleus of a Eukaryotic Cell (9)
Histone and DNA modification is one mechanism to alter the character of nucleosomes.
DNA and histones are held together by noncovalent bonds.

38. The Nucleus of a Eukaryotic Cell (13)
The histone code hypothesis states that the activity of a chromatin region depends on the degree of chemical modification of histone tails.
Histone tail modifications influence chromatin in two ways:
Serve as docking sites to recruit nonhistone proteins.
Alter the way in which histones of neighboring nucleosomes interact with one another.

39. Methyl groups can be added to the DNA Specificity: Cytosines 5’-CG-3’
-----DNA metylation
Methyl groups can be added to the DNA
Specificity: Cytosines
5’-CG-3’
Enzyme: methyl transferase
Function: gene inactivation
Epigenetic changes (i.e., function)----Epigenomic

40. -----Histone acetylation
Acetyl groups can be added to the Histones
Specificity: H3 and H4
Enzyme: Acetyl transferase
Function: gene activation
The prefect combination for gene inactivation
DNA methylation
H3/H4 de-acetylation and metylation
(heterochromatin)
Heterochromatin protein (HP)1

41. HDAC: histone De-acetylase complex HAT: histone acetyltransferase

42. The Nucleus of a Eukaryotic Cell (16)
Telomeres
The end of each chromosome is called a telomere and is distinguished by a set of repeated sequences.
New repeats are added by a telomerase, a reverse transcriptase that synthesizes DNA from a RNA template.
Telomeres are required for the complete replication of the chromosome because they protect the ends from being degraded.
Telomerase activity is thought to have major effects on cell life

43. Telomeres

44. The role of telomerase

45. The Nucleus of a Eukaryotic Cell (22)
The Nuclear Matrix
The nuclear matrix is a network of protein-containing fibrils.
It serves as more than a skeleton to maintain the shape of the nucleus and anchoring the machinery involved in nuclear activities.

46. Control of Gene Expression in Eukaryotes (2)
Genes are turned on and off as a result of interaction with regulatory proteins.
Each cell type contains a unique set of proteins.
Regulation of gene expression occurs on three levels:
-Eukaryotes
---Genomic control
---Transcriptional control
Transcriptional-level control
Processing-level control
---Posttranscriptional control

47. ---Transcriptional control
-----differential gene transcription
-----control elements
(transcriptional
factors)

48. 12.4 Transcriptional-level control (1)
1. Differential transcription is the most important mechanism by which eukaryotic cells determine which proteins are synthesized.

49. Transcriptional-level control (2)
2. DNA microarrays can monitor the expression of thousands of genes simultaneously. a) Immobilized fragments of DNA are hybridized with fluorescent cDNAs. b) Genes that are expressed show up as fluorescent spots on immobilized genes. c) Microarrays a provide a visual picture of gene expression.

50. The construction of a DNA microarray
(DNA chip or biochip)

51. Demonstration of Differential Transcription by Nuclear Run-on Transcription Assays

52. Using a DNA Microarray to Profile the Patterns of Gene Expression in Two Different Cell Types

53. Transcriptional-level control (3)
The Role of Transcription Factors in Regulating Gene Expression
Transcription factors are the proteins that either acts as transcription activators or transcription inhibitors.
A single gene can be controlled by different regulatory proteins.
A single DNA-binding protein may control the expression of many different genes.

54. Transcriptional-level control (5)
The Structure of Transcription Factors
Transcription factors contain a DNA-binding domain and an activation domain.

55. Transcriptional-level control (6)

56. -----control elements
Anatomy of a Typical Eukaryotic Gene, with Its Core Promoter and Proximal Control Region
-----control elements
Enhancer
Silencer
monocistronic

57. Model for Enhancer Action
Suppressor or silencer
DHAC: De-acetylase complex
MTC: Methyl transferase complex
Co-repressor
HAT: histone acetyl transferase
Co-activator
Remodeling complex

58. Combinatorial Model for Gene Expression

59. -Eukaryotes
---Genomic control
---Transcriptional control
---Post-transcriptional control
----alternative splicing
----translational
----degradation of mRNA
----protein degradation

60. Typical mammalian gene has 7-8 exons over ~16 kb
Exons are short (~ bp)
Introns are long (~>1kb)
Pre-mRNA
mRNA
Splicing occurs in the nucleus
Figure 26.2
Eukaryotic genes often contain intervening sequencings (introns) that separate the exons and must be removed for proper protein translation to occur.
Removal of introns involves a complicated “molecular device” called a spliceosome that involves protein and RNA (like a ribosome).
Splicing allows an increase in the information content of a genome due to alternative splicing products that produce novel proteins.

61. Nuclear Splice Junctions Are Short Sequences
Splice sites are the sequences immediately surrounding the exon–intron boundaries.
They are named for their positions relative to the intron.

62. The 5′ splice site at the 5′ (left) end of the intron includes the consensus sequence GU.
The 3′ splice site at the 3′ (right) end of the intron includes the consensus sequence AG.
The GU-AG rule describes the requirement for these constant dinucleotides at the first two and last two positions of introns in pre-mRNAs.
Splice sites are generic.
GT…AG (DNA)-(~98 %);(~2% ?)
Directional (one direction)
The apparatus for splicing is not tissue specific (with some exceptions).
The mechanism by which pairs of splice sites are correctly identified is unknown.
Figure 26.3

63. 12.7 Post-translational Control: Determining Protein Stability (1)
The factors that control a protein’s lifetime are not well understood.
Protein stability may be determined by the amino acids on the N-terminus.
Degradation of proteins is carried out within hollow, cylindrical proteasomes.

64. Post-translational Control: Determining Protein Stability (2)
Proteasomes recognize proteins linked to ubiquitin ( small protein)
Ubquitin is transferred by ubiquitin ligases to proteins being degraded.
Once polubiquitanated, a protein is recognized by the cap of the proteasome.
Once degraded, the component amino acids are released back into the cytosol.

65. Proteasome structure

67. Regulatory RNA

68. A Few Approaches to the Study of Cytomembranes (6)
Insights from Studying Mutants (continued)
RNA interference is a process in which cells produce small interference RNAs (siRNAs) that bind to specific mRNAs and inhibit the translation of these into proteins.
Scientists can identify genes involved in a particular process by determining which siRNAs interfere with that process.

69. Inhibition of gene expression with RNA interference

70. What is RNA interference?
Shooting down mRNA

71. Plasmid
Virus

72. The Central Dogma of Molecular Biology
The flow of information is DNA  RNA protein.
Some viruses can use RNA as a template for the synthesis of DNA in reverse transcription
Many DNA fragments do not encode polypeptides; their end-products are RNA molecules !
© John Wiley & Sons, Inc. 72

73. Unannotaed (?) polyA RNA, polysomal RNA, lincRNA, miRNA, piRNA, siRNA, tmRNA, SmY RNA, scaRNA, gRNA, aRNA, crRNA, tasiRNA, rasiRNA, 7SK RNA

75. Bill Douherty and Lindbo 1993 Hamilton and Baulcombe 1998
Jorgensen 1990
van der Krol 1990
Gene injection (pigmentation
Enzyme-petunias)
Expectation: more red color
Co-suppression of transgene
and endogenous gene.
Bill Douherty and Lindbo 1993
Hamilton and Baulcombe 1998
Gene injection with a complete tobacco
etch virus particle.
Expectation: virus expression
Co-suppression of transgene
and virus particles via RNA.
Identification of short antisense RNA
sequences
dsRNA?
How?
Ambros 1993 (2000)
Fire and Mello 1998
Identification of small RNA in
C. elegans (micro RNA)
Injection of dsRNA into C. elegans
RNA interference (RNAi) or silencing

76. Shooting mRNA means RNA interference

77. What is RNA interference?
--Gene “knockdown”
--A cellular mechanism that degrades unwanted RNAs in the cytoplasm but not in the nucleus. Why?
--A way for the cell to defend itself.

78. The RNAi process RNA interference: --A type of gene regulation
--Involve small RNA molecules
--Induce a double stranded RNA
The RNAi process

79. Step 1 dsRNA is processed into sense and antisense RNAs
21-25 nucleotides in length
have 2-3 nt 3’ overhanging ends
Done by Dicer (an RNase III-type enzyme)

80. Step 2 The siRNAs associate with RISC (RNA- induced silencing
complex) and
unwind

81. Step 3
the antisense siRNAs act as guides for RISC to associate with complimentary single-stranded mRNAs.

82. Step 4
RISC cuts the mRNA approximately in the middle of the region paired with the siRNA
The mRNA is degraded further

84. Catalysis: RdRP copies RNA making more ds RNA.
ssRNA (exogenous)
RNA-dependent
RNA polymerase
Catalysis: RdRP copies
RNA making more ds RNA.
Dicer complex: RNAase III
with ATP hydrolysis
requirement.
Dicer cuts, unwinds dsRNA
and generates more siRNA.
More RdRP is activated and
more dsRNA is made.
RISC complex:RNA-Inducible
Silencing Complex with ATP
hydrolysis.
(RdRP)
(endogenous)

85. Gene regulation by small RNAs
Dicer gene in C. elegans
siRNAs degrade mRNA
to stop gene expression
quickly
Small temporal (St) RNAs
prevent translation to
stop gene expression quickly

86. -your RNAi?
--MicroRNAs (miRNA) are single-stranded RNA molecules of about nucleotides in length,
which regulate gene expression (down-regulation).
--miRNAs are encoded by genes that are non-coding RNAs ( no proteins are made)
--Stem-loop or hairpin loop intra-molecular base pairing is a pattern that can occur in
single-stranded DNA or, more commonly, in RNA.

87. Philadelphia chromosome
Chronic myelogenous leukemia (CML)
--translocation of genetic
(between chromosome 9 and chromosome 22, and contains a fusion gene called BCR-ABL)
ABL: tyrosine kinase
BCR: substrate
ATGCGTAAGGTGCC
RNAi