The Yeast nRNAP II

• Has 12 subunits, based on traditional enzyme purification and epitope tagging.

• Gene knockouts indicate that 10 subunits are essential, & 2 are required under

certain conditions (4 and 9). • The 2 large subunits (genes Rpb1 and Rpb2)

have regions of homology with the and ’ subunits of the E. coli RNAP and seem to function similarly.

• RPB1 is responsible for -aminitin sensitivity.

Epitope tagging

1. Add “tag” (small peptide) to a subunit gene.

2. Transform in tagged gene.3. Use a specific antibody for

the tag to “immunoselect” the tagged protein.

4. Other proteins that come down with the tagged protein are detected by SDS-polyacrylamide gel electrophoresis.

Fig. 10.6

RNAP II purified using the epitope tag on Rpb3; has 10 subunits.

RNAP II purified from wild-type yeast: 12 subunits.

Rpb1 subunit is phosphorylated.

Fig. 10.71990

1992

nRNAP II Heterogeneity

• Largest subunit - Subunit II (Rpb1 gene) is phosphorylated on carboxyl-terminal domain (CTD).

• 3 forms of this subunitIIa - primary product IIb - derived from IIa by removal of CTD

(artifact of purification)IIo - phosphorylated form of IIa

• Functionally different: IIa-enzyme binds promoter; IIo-enzyme is in elongation phase.

Fig. 10.10

Structure of yeast RNAP II lacking Rbp4 and 7

Fig. 10.11

Surface structure of yeast RNAP II

Mg2+ at active site - pink sphere

Fig. 10.12

Model for the path of a straight DNA in the initiation complex

Clamp opens to let DNA template have access.

Figure 10.13

Structure of the elongation complex.

Fig. 10.14

Figure 10.14

Synthetic DNA used to obtain a mimic of a transcribing complex after

translocation

Fig 10.17

Dideoxynucleotided

Fig. 10.18Matched and Mismatched Nucleotides in A (active) and

E (entry) sites

Fig 10.18

Promoters for the 3 Nuclear Promoters for the 3 Nuclear RNA polymerases (nRNAPs)RNA polymerases (nRNAPs)

Order of topics:

1. Class II promoters (for nRNAP II)

2. Class I promoters (for nRNAP I)

3. Class III promoters (for nRNAP III)

4. Enhancers and Silencers

Generic Class II Promoter1. Upstream element2. TFIIB recognition element3. TATA Box (at approx. –25)4. Initiator 5. Downstream promoter element (can substitute for

TATA box)

Promoters may lack one or more of these and still function.

1. 2. 3. 4. 5.

Figure 10.20

TATA Box of Class II PromotersTATA Box of Class II Promoters

• TATA box = TATAAAA

• Defines where transcription starts.

• Also required for efficient transcription for some promoters.

• Some class II promoters don’t have a TATA box.

Effects of Deletions in the SV40 Early Promoter on the Transcription Start Site

(b) Mapping of the transcription start site on the

mutant DNAs by S1 nuclease digestion. extension.Fig 10.21

S1 mapping of the 5’ end of a RNA Transcript

A 5’ end-labeled single-stranded DNA probe is prepared from the template strand. After hybridization to RNA and digestion with S1, the size of the protected DNA indicates approx. how far to the RNA 5’-end.

Similar to Fig. 5.26

High resolution analysis of the 5’-end of an RNA transcript by primer extension.

Primer is an end-labeled DNA oligonucleotide (~20 nt) that is complementary to a sequence in the RNA ~150 nt from the expected 5’ end.

Lane E- extended DNA product

Lanes A,C, G, T – sequence ladder generated with the same oligo primer, but on the corresponding cloned DNA.

From Fig. 5.29

Linker scanning mutagenesis of a stretch of DNA.

Replace ~10 bp of natural sequence with 10 bp of synthetic DNA.

Do this periodically throughout the stretch of DNA you want to examine for important sequences.

From Fig. 10.22

Linker scanning mutagenesis of the Herpes virus tk promoter identifies 2 important upstream regions.

DNA was injected into frog oocytes, and the transcribed RNA analyzed by primer extension.

Fig. 10.23

Regions -47 to -61 and -80 to -105 contain GC boxes (GGGCGG and CCGCCC).

Upstream Elements of Class IIUpstream Elements of Class II

• Can be several of these, two that are often found:

1. GC boxes (GGGCGG and CCGCCCC)– Stimulate transcription in either orientation– May be multiple copies– Must be close to TATA box (different from enhancers)– Bind the Sp1 factor

2. CCAAT box– Stimulates transcription– Binds CCAAT-binding transcription factor (CTF) or

CCAAT/enhancer-binding protein (C/EBP)

Class I Promoters (for nRNAP I)Class I Promoters (for nRNAP I)

• Sequences less conserved than Class II• Usually 2 parts:

– UCE : upstream control element , -150 to -100 in human rRNA

– Core: from - 45 to +20

• Spacing between elements also important

-150 -100 -50 +20

UCE Core

Results of Linker Scanning mutagenesis of the human rRNA promoter.

The DNA was transcribed in vitro, and the efficiency is expressed relative to the wild-type promoter.

Fig. 10.24

Class III promoters (for nRNAP III)Class III promoters (for nRNAP III)

• 3 types:1. Two (Types I and II) have Internal promoters

- 5S rRNA (Box A, Intermediate element, Box C)- tRNA (Box A, B)

2. Type III is upstream of coding region- e.g., human U6 RNA- contain TATA box and resemble class II promoters

Effect of deletions at the 5’-end of the Xenopus 5S rRNA gene on its transcription in vitro.

Result: It required deleting more than 50 bp into this small gene to knock out its transcription.

Conclusion: the promoter for the 5S rRNA gene is internal!

Numbers at bottom of lanes are the bp deleted.

Fig. 10.27

Enhancers and SilencersEnhancers and Silencers

• Enhancers stimulate transcription, silencers inhibit

• Both are orientation independent– Flip 180 degrees, still work

• Both are position independent– Can work at a distance from promoter

• Bind regulated (tissue-specific) transcription factors

An enhancer in an intron of the An enhancer in an intron of the γγ2b 2b gamma-globulin gamma-globulin gene.gene.

(a) Genes were constructed with the enhancer inverted (B), or moved upstream of the gene (C) and inverted (D). The DNAs were transfected into mouse cells and synthesis of the protein was assessed by pulse-

labeling with a radioactive amino acid and immunoprecipitation.

Fig. 10.30