Plant Nuclear Gene Expression & Regulation

A lot of steps to regulate:

1. Transcription*

2. Capping

3. 3' maturation, cleavage & polyadenylation

4. Splicing*

5. Transport to Cytoplasm

6. Stabilization/Destabilization of mRNA*

7. Translation*

* have the most regulation.

Likely order of events in producing a mature mRNA from a pre-mRNA.

Transcription: 3 DNA-Dependent RNA Polymerases

1. Pol I - synthesizes 45S rRNA precursor, found in nucleoli (45S18S, 28S, 5.8S rRNAs)

[S refers to rate of sedimentation (Fig. 6.33 in Buchanan), approx. equivalent to size of macromolecule]

2. Pol II - synthesizes mRNA precursors, some snRNAs

3. Pol III- synthesizes 5S rRNAs, tRNAs, small nuclear RNAs (snRNAs)

All 3 polymerases are multi-subunit; have some large, unique subunits; and 5 small, shared subunits (at least in yeast).

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)

RNA Polymerase II

1. 2 large subunits have regions of homology with ß and ß’ subunits of E. coli RNAP.

2. Largest subunit is phosphorylated on its COOH-terminal domain (CTD)– Phosphor. needed for transition from initiation

elongation– CTD also interacts with other proteins

3. Does not bind DNA by itself, requires other proteins to bind promoter first!

Fig. 6.30, Buchanan et al.

TFII – transcription factors for RNA Pol II

RNAPII – RNA Pol II

RNAP II Promoters

• Class-II promoters have 4 components:1. Upstream element(s)2. TATA Box (at approx. –25)3. Initiation region (includes the first transcribed nt, +1)

4. Downstream element

Many class II promoters lack 3 and 4; a few lack 2.

1. 2. 3. 4.

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)

Upstream elements: Class II promoters

Found in many class II promoters:

1. GC boxes (GGGCGG and CCGCCCC)– Stimulate transcription in either orientation– May be multiple copies– Must be close to TATA box

2. CCAAT box

– Stimulates transcription– Binds CTF (Cat-box transcription factor)

Enhancers and Silencers

1. Enhancers stimulate transcription, while Silencers inhibit.

2. Orientation-independent– Flip 180 degrees, still work

3. Position-independent (mostly) – Can work at a distance from promoter core– Enhancers have been found all over

4. Bind regulatory transcription factors

Transcription factors for Class II promoters

1. Basal factors: required for initiation at most promoters; interact with TATA box.

2. Upstream factors: bind common (consensus) elements upstream of TATA, including proximal-promoter elements (e.g., CCAAT box); increase efficiency of initiation.

3. Inducible (regulated) factors: work like upstream factors but are regulatory (produced or active only at specific times/tissues); interact with enhancers or silencers.

Assembly of the RNA Pol II Initiation Complex= basal factors + RNAP II

Fig. 7.45, Buchanan et al.

TFIIF delivers Pol II

TFIIH PO4ylates the LS of Pol II, allowing it to escape the promoter.

Eukaryotic Transcription Factors: Structure

• Mostly about factors that bind USEs:

1. Modular structure:– DNA-binding domain– Transcription-activating domain

2. Can have > 1 of each type of module 3. Many factors also have a dimerization

domain (some can form heterodimers).

DNA-binding domains

1. Zinc – containing modules2. Homeodomains (conserved amino acid seq.)

3. bZIP and bHLH motifs

4. AP2 (mainly in plants)

(not an exhaustive list, just what might be on the test!)

Activation from a Distance: Enhancers

• 3 possible models

Factor binding induces:

1. Supercoiling of the promoter DNA

2. Sliding of the complex to the promoter

3. Looping out of DNA between enhancer and promoter

3 Models of possible enhancer action.

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:

1. 4 different histones in the core (H2a, H2b, H3, H4 x 2 = octamer)

2. 146 bp of DNA wrapped around core

3. Histone H1 on outside

H2B

H4

H3

H1

H2B

H2A

DNA

Packing ratio ~5

Nucleosome core = octamer of histones (2 each of H2A, H2B, H3, H4) + 2 wraps (145 bp) of DNA

Fig. 7.49 Buchanan et al.

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.

Histones can be modified (for chromatin remodeling)

In Vivo Studies • Promoters of active genes are often

deficient in nucleosomes

SV40 virus minichromosomes with a nucleosome-free zone at its twin promoters.

Fig. 13.25

Can also be shown for cellular genes by DNase I digestion of chromatin – promoter regions are hypersensitive to DNase I.

Post-Transcriptional Processes

1. Capping

2. 3’ end formation (not much regulation of the above

steps)

3. Splicing – alternative splicing

4. Translation – regulate initiation step

Cap Functions

• Capping also includes methylation of the ribose (2-OH) on nt #1 and sometimes #2.

• Cap functions:

1. Protection from 5 exoribonucleases

2. Enhances translation in the cytoplasm

3. Enhances transport from the nucleus

4. Enhances splicing of the first intron (for some pre-mRNAs)

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

3' End Formation

CIS (elements)• AAUAA is the key signal in higher plants, its

found ~20 nt from the polyA-tail.– Other sequences 5' to the AAUAA also

important.

TRANS (factors)• 3' end formation requires at least:

– an endonuclease & recognition factors– a poly(A) polymerase (PAP)– a poly A-binding protein (PAB)