1. Alternate sigma factor usage: controls selective transcription
Ways to Regulate Transcription
Alternate sigma factor usage: controls selective transcription
of entire sets of genes
vegetative
(principal s)
s70 +1
צמיחה
(16-19 bp)
(5-9 bp)
TTGACA
TATAAT A
heat shock +1
s32
(13-15 bp)
(5-9 bp)
CNCTTGA
CCCATNT A +1
nitrogen
starvation
s60
(6 bp)
(5-9 bp)
CTGGNA
TTGCA A
התרגיל ביום חמישי זה – !

2. טרנסקריפציה באוקריוטים

5. The pathway of gene expression

6. Pre-mRNA
* microRNAs (miRNA) RNA molecules of about 21–23 nucleotides in length, which regulate gene expression. are processed from primary transcripts known as pri-miRNA.

8. 5’UTR
3’UTR

9. בתאי כבד בעכבר

10. PolyA RNA in the nucleus and cytoplasm in two human cell lines

12. Eukaryotic Promoters
More complex and diverse than prokaryotic promoters
RNA polymerase I promoters
Multiple copies of rRNA genes exist
RNA polymerase I recognizes only one species-specific promoter
Requires a core promoter element and an upstream promoter element
RNA polymerase III promoters have variable locations relative to the transcribed gene
More attention has been paid to RNA polymerase II promoters because they are involved in the transcription of mRNA

13. RNA polymerase

14. מיקום +1
רמה נמוכה (בזלית)
יעילות ותדירות
אינטרונים E S RE

18. אין אחידות במיקום וברצפי הבקרה בין גנים שונים.

21. שלבי הטרנסקריפציה
Initiation
Elongation
Termination

23. TATA-Binding Protein (TBP)
Formation of the preinitiation complex (PIC) begins when TBP protein of TFIID binds the TATA box of a promotor
DNA binds the concave surface in a sharply bent conformation
Double helix is partially unwound and minor groove widens

26. TFII = transcription factor of RNA PolII
ע"מ שRNA פולימראז יצליח להיקשר לאזור הפרומוטר TF צריכים להיקשר.

27. תהליך
ה-INITIATION

28. Initiation complex

31. קומפלקס פרה-איניציציה
יציבות ותדירות
יצירת קומפלקס זה
קובעת את כמות וקצב
סינטזת ה-RNA

33. Expanding the functions of RNA polymerase

34. אופן אקטיבציה של טרנסקריפציה בתאים אוקריוטים

35. Stages in Initiation of Transcription
Bacterial transcription
Closed complex: holoenzyme+promoter
Open complex (DNA melting, not need ATP)
Abortive transcripton
Productive initiation
Transcribe past +9
Sigma dissociates
Elongation
Eukaryotic transcription
Preinitiation complex (PIC) assembly
PIC activation (DNA melting, needs ATP)
Abortive transcription
Productive initiation
CTD phosphorylated
Promoter clearance
Elongation

36. Structure of RNA Polymerase II
RNA polymerase II from yeast has been extensively characterized
Contains two large subunits that are homologs of prokaryotic RNA polymerase subunits b and b’
Contains 10 smaller subunits, including homologs of a and r
Structural features
Thumb
DNA-binding channel
Channel for single-stranded RNA

37. 3-dimensional view of yeast RNA Pol II
Core
Holoenzyme
Both yeast RNA Pol II and E. coli RNA polymerase core
Have a similar shape and have the channel for DNA template.
RNA polymerase II from yeast has been extensively characterized
Contains two large subunits that are homologs of prokaryotic RNA polymerase subunits b and b’
Contains 10 smaller subunits, including homologs of a and r
Structural features
- Thumb
- DNA-binding channel
- Channel for single-stranded RNA

38. Parallels between initiation pathway in prokaryotes and eukaryotes
From Eick et al. (1994)
Trends in Genetics 10:

39. Initiation: Assembly of the PIC
TBP component of TFIID binds the TATA box
TFIIA and TFIIB bind
TFIIF binds RNA polymerase and escorts it to the complex
TFIIE and TFIIF complete the PIC

40. initiation

41. Prokaryotes: RNA polymerase Binds to promoter Pulls 2 strands apart
INITIATION
Prokaryotes: RNA polymerase
Binds to promoter
Pulls 2 strands apart
Eukaryotes: Transcription initiation complex
Transcription factors
Proteins
Bind to the promoter
RNA polymerase

42. Elongation
NTPs

43. Elongation

44. RNA polymerase II

45. TERMINATION RNA polymerase meets the terminator
Terminator sequence: AAUAAA
RNA polymerase releases from DNA
Prokaryotes-releases at termination signal
Eukaryotes-releases base pairs after termination signal

46. polyadenylation

49. Initiation, Elongation, and Termination
Helicase activity of TFIIF promotes DNA unwinding
Kinase activity of TFIIF phosphorylates RNA polymerase, causing a conformational change in the polymerase that initiates transcription
TFIIA, TFIIB, and TFIID (including TBP) remain bound to the promoter to aid in PIC reassembly
TFIIH and TFIIE are released
Activity of polymerase is enhanced by proteins called elongation factors
Termination mechanism is not well understood
Polymerase is dephosphorylated and released

50. Enhancers and Silencers
The activities of many promoters in eukaryotes are increased by enhancers and decreased by silencers
Can be upstream, downstream, or in the midst of a transcribed gene
Activators or repressors can bind to enhancers and silencers to influence RNA polymerase binding to promoters
Enhancers and silencers act only in cells that contain the appropriate activator or repressor proteins

51. Transcription at Other Promoters
RNA polymerase II promoters that lack TATA boxes require several of the same transcription factors that initiate transcription from TATA boxes, including TBP
Transcription by RNA polymerase I and RNA polymerase III requires different sets of transcription factors, but TBP is required in all cases
Studying transcription initiation is difficult because transcription factors are present at very low concentrations

52. TBP is used by all 3 RNA polymerases
TBP is a subunit of an important GTF for each of the 3 RNA polymerases:
TBP or TFIID for Pol II
SL1 for Pol I
TFIIIB for Pol III
It does NOT always bind to TATA boxes; promoters for RNA Pol I and Pol III (and even some for Pol II) do not have TATA boxes, but TBP is still used.
The GTFs that contain TBP may serve as positioning factors for their respective polymerases.

53. RNA Pol I

54. Signal transduction of Pol I

60. Transcription complex

61. קומפלקס האלונגציה

62. transcription

64. polyA-PolII termination

66. Transcription regulation

69. Upstream control element
Upstream binding factor
Transcription associated factor

72. 30 ח. אמינו
2-30 אתרי קישור
נקשרים למיגור גרוו
משמשים כTF
ציסטאין
היסטידין

79. Gene expression

81. Basics of cell biology:development
Fertilized egg

82. Basics of cell biology:development
Cells differentiation
is due to different gene
Switches going on and off
These cells are different
because they express a
subset of their 24,000 genes

83. Gene
Promoter
To specify a new cell: place it in the right environment or

84. EGF
Hedgehog
Wnt
Gene
Promoter
Retinoic acid
FGF
HGF
To specify a new cell: place it in the right environment or
Substitute drugs/proteins for environment
Mophogens

85. Chromosome localization in interphase
In interphase, chromosomes appear
to be localized to a sub-region of the
nucleus.

86. Domain opening is associated with movement to non-hetero-chromatic regions

87. Proposed sequence for activation
1. Open a chromatin domain
Relocate away from pericentromeric heterochromatin
Establish a locus-wide open chromatin configuration
General histone hyperacetylation
DNase I sensitivity
2. Activate transcription
Local hyperacetylation of histone H3
Promoter activation to initiate and elongate transcription

88. From silenced to open chromatin

89. Movement from hetero- to euchromatin

90. Nucleosoe remodelers and HATs further open chromatin

91. Assembly of preinitiatin complex on open chromatin

92. Transcription factor binding to DNA is inhibited within nucleosomes
Affinity of transcription factor for its binding site on DNA is decreased when the DNA is reconstituted into nucleosomes
Extent of inhibition is dependent on:
Location of the binding site within the nucleosome.
binding sites at the edge are more accessible than the center
The type of DNA binding domain.
Zn fingers bind more easily than bHLH domains.

93. Stimulate binding of transcription factors to nucleosomes
Cooperative binding of multiple factors.
The presence of histone chaperone proteins which can compete H2A/H2B dimers from the octamer.
Acetylation of the N-terminal tails of the core histones
Nucleosome disruption by ATP-dependent remodeling complexes.

94. Binding of transcription factors can destabilize nucleosomes
Destabilize histone/DNA interactions.
Bound transcription factors can thus participate in nucleosome displacement and/or rearrangement.
Provides sequence specificity to the formation of DNAse hypersensitive sites.
DNAse hypersensitive sites may be
nucleosome free regions or
factor bound, remodeled nucleosomes which have an increased accessibility to nucleases.

95. Chromatin remodeling ATPases catalyze stable alteration of the nucleosome
II: form a stably remodeled dimer, altered DNAse digestion pattern
III: transfer a histone octamer to a different DNA fragment

96. Covalent modification of histone tails
N-ARTKQTARKSTGGKAPRKQLATKAARKSAP...- H3 4
9 10 14 18 23
27 28
N-SGRGKGGKGLGKGGAKRHRKVLRDNIQGIT...- H4 1 5 8 12 16 20
phosphorylation
acetylation
methylation

97. Yeast SAGA interacting with chromatin