Plant Nuclear Gene Expression

1
Plant Nuclear Gene Expression Regulation

• A lot of steps to regulate
• Transcription
• Capping
• 3' maturation, cleavage polyadenylation
• Splicing
• Transport to Cytoplasm
• Stabilization/Destabilization of mRNA
• Translation

2
Order of events in producing a mature mRNA from a
pre-mRNA with 1 intron.
3
Transcription 3 DNA-Dependent RNA Polymerases

• Pol I - synthesizes 45S rRNA precursor, found in
  nucleoli (45S?18S, 28S, 5.8S rRNAs)
• S refers to rate of sedimentation (Fig. 6.33
  in Buchanan), approx. equivalent to size of
  macromolecule
• Pol II - synthesizes mRNA precursors, some
  snRNAs
• 3. Pol III- synthesizes 5S rRNAs, tRNAs, small
  nuclear RNAs (snRNAs)
• All 3 polymerases are multi-subunit, with some
  large unique subunits and 5 small shared subunits
  (in yeast).

4
Relative cellular RNA abundance

• Ribosomal RNAs (rRNAs) 90
• Transfer RNAs (tRNAs) 5
• Messenger RNAs (mRNAs) 2
• The rest (3)
• Signal recognition particle (SRP) RNA
• Small nuclear RNAs (snRNAs)
• Small nucleolar RNAs (snoRNAs)
• Micro RNAs (miRNAs)

5
RNA Polymerase II

• 2 large subunits have regions of homology with ß
  and ß subunits of E. coli RNAP.
• Largest subunit is phosphorylated on
  COOH- terminal domain (CTD)
• Phosphor. needed for transition from initiation ?
  elongation
• CTD also interacts with other proteins
• These RNAPs (I-III) do not bind DNA specifically
  by themselves, require other proteins to bind
  promoter first!

6
TFII transcription factors for RNA Pol
II RNAPII RNA Pol II
Fig. 6.30, Buchanan et al.
7
RNAP II Promoters

• Class-II promoters have 4 components
• Upstream element(s)
• TATA Box (at approx. 25)
• Initiation region or start site (1)
• Downstream element

1. 2. 3. 4.
Many class II promoters lack 3 and 4. Some lack 2.
8
TATA Box of Class II Promoters

• TATA box TATAAAA
• Defines where transcription starts
• Also required for efficient transcription for
  some promoters
• Bound by TBP TATA box binding protein (in
  complexes like TFIID)

9
Upstream Elements of Class II Promoters

• Found in many class II promoters
• GC boxes (GGGCGG and CCGCCCC)
• Stimulate transcription in either orientation
• May be multiple copies
• Must be close to TATA box (enhancers dont)
• CCAAT box
• Stimulates transcription
• Binds CCAAT-binding transcrip. factor (CTF)
• Also tend to be close to the TATA box

10
Enhancers and Silencers (and insulators)

• Enhancers stimulate transcription, Silencers
  inhibit
• Both are orientation-independent
• Flip 180 degrees, still work
• Both are somewhat position-independent
• Can work at a distance from promoter
• Enhancers have been found all over
• Bind regulated transcription factors

11
Transcription Factors for Class II Promoters

• Basal factors required for initiation at most
  promoters interact with TATA box.
• Upstream factors recognize consensus elements
  upstream of TATA box ubiquitous increase
  efficiency of initiation. Interact with proximal
  promoter elements (CCAAT box).
• Inducible (regulated) factors Work like
  upstream factors but are regulatory made or
  active only at specific times or tissues.
  Interact with enhancers or silencers.

12
Assembly of the RNA Pol II Initiation Complex
basal factors RNAP II
Fig. 7.45, Buchanan et al.
13
Eukaryotic Transcription Factors Structure

• Refers mostly to factors that bind upstream
  elements (USE)
• Modular structure
• DNA-binding domain
• Transcription-activating domain
• Can have gt 1 of each type of module
• Many factors also have a dimerization domain
  (some can even form heterodimers).

14
DNA-binding domains

• More easily recognized and understood, compared
  to activation domains
• Zinc containing modules
• Homeodomains
• bZIP and bHLH motifs
• AP2 (mainly in plants), partly evolved from an
  HNH endonuclease

15
GAL4-DNA Complex

• DNA-binding domain
• 2 Zn2 bound by 6 cysteines
• A Short a helix that docks into major groove

Dimerization domain - Coiled coil (a helices)
Fig. 12.4
16
Leucine Zipper

• helices form a coiled-coil with interdigitating
  leucines.

17
Activation from a Distance Enhancers

• 3 possible models
• Factor binding induces
• Supercoiling of the promoter DNA
• Sliding of the complex to the promoter
• Looping out of DNA between enhancer and promoter

18
P box minimal promoter Yellow oval TFIID Red
oval RNAPII core
3 Models of possible enhancer action.
19
Chromatin Modification

• Transcription can also be regulated by modifying
  chromatin (histones) highly transcribed genes
  have less condensed chromatin.
• Basic unit of chromatin is the nucleosome
• 4 different histones in the core (H2a, H2b, H3,
  H4 x 2 octamer)
• 146 bp of DNA wrapped around core
• Histone H1 on outside

20
Nucleosome core octamer of histones (2 each of
H2A, H2B, H3, H4) 2 wraps (or 145 bp) of DNA
Packing ratio 5
21
Histones can be modified (for chromatin
remodeling)
Histone acetylation (right) causes localized
unpacking of nucleosomes, which enhances factor
binding to DNA. De-acetylated histones (left)
bind DNA more strongly, and the nucleosomes
condense into a solenoid this inhibits factor
binding to DNA targets.
Fig. 7.49 Buchanan et al.
22
In Vivo Studies

• Promoters of active genes are often deficient in
  nucleosomes

SV40 virus minichromosomes with a
nucleosome-free zone at its twin promoters.
Can also be shown for cellular genes by DNase I
digestion of chromatin promoter regions are
hypersensitive to DNase I.
Fig. 13.25
23
Post-Transcriptional Processes

• Capping
• 3 end formation
  (not much regulation of the above steps)
• Splicing alternative splicing
• Translation regulate initiation step

24
Cap Functions

• Capping can also include methylation of the 2?-OH
  on the ribose of the 1st or 2nd transcribed
  nucleotides
• Cap functions
• Protection from 5? exoribonucleases
• Enhance translation in cytoplasm
• Enhance transport from the nucleus
• Enhance splicing of the first intron for some
  pre-mRNAs.

25
3-end Processing Polyadenylation Mechanism

• Transcription extends beyond mRNA end
• Transcript is cut at 3 end of what will become
  the mRNA
• PolyA Polymerase adds 250 As to 3 end
• Extra RNA degraded

26
3' End Formation

• AAUAA is a signal for 3' end formation in higher
  plants, occurs 20 nt 5' to the polyA-tail
• Other sequences 5' to the AAUAA also important
• 3' end formation requires at least
• an endonuclease recognition factors
• a poly(A) polymerase (PAP)
• a poly A-binding protein (PAB)