Eukaryotic Transcription.... Similar Themes, But a Little Different

Chapters 19, 20 & 21

Initiation Is Followed by Promoter Clearance and Elongation

• The histone octamers must be temporarily modified around the promoter sequences when RNA polymerases are bound during the transition of the RNA polymerase through the DNA.

• The CTD coordinates processing of RNA with transcription – the CTD contains heptapeptide repeat sequences -/YSPTSPS/YSPTSPS/YSPTSPS/- – Phosphorylation of the CTD is required for promoter clearance and elongation to begin – Further phosphorylation of the CTD is required at some promoters to end abortive

initiation. – DNA-directed RNA polymerase II subunit RPB1 - an enzyme that in humans is encoded by the

POLR2A gene. RPB1 is the largest subunit of RNA polymerase II. It contains a carboxy terminal domain (CTD) composed of up to 52 heptapeptide repeats (YSPTSPS), ONLY 26 heptapetide repeats in Yeast

• Transcribed genes are preferentially repaired when DNA damage occurs.

• TFIIH also provides the link to a complex of repair enzymes.

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• An enhancer activates the promoter nearest to itself, and can be any distance either upstream or downstream of the promoter.

• A UAS (upstream activating sequence) in yeast behaves like an enhancer, but works only upstream of the promoter… not so rigorous in other eukaryotes.

Enhancer action is independent of its location

RNA polymerase exists as a holoenzyme

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• (a) The HU protein dimer complexed with DNA

• (b) Binding of an E. coli IHF dimer to DNA induces a 180° Turn

• (c) Structure of the N-terminal domain of E. coli H-NS dimer.

All structures show protein secondary structures and tubular DNA

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Architectural proteins control the structure of DNA

•Activators bound to a promoter can “recruit” basal transcription factors. •Alternatively, bound activators may trans-activate basal TFll components already bound near to the start site.

•Different associated factors can be present in the basal transcription complex at different promoters.

Just like CRP and LacI regulatory proteins in prokaryotes… Bipartite nature of transcription factors (activators) have separate DNA binding and activator domains

General types of interaction domains in regulatory activator proteins (TF’s etc)

• Helix-Loop-Helix motif facilitates protein-protein interaction. A separate basic region interacts with DNA.

• Leucine zipper motif facilitates protein-protein interaction. A separate basic region interacts with DNA.

• Homeodomain motif facilitates DNA binding • Zinc finger motif can facilitate DNA binding or protein

interaction

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The λ repressor of bacteriophage lambda employs a helix-turn-helix (left; green) to bind DNA (right; blue and red).

FIGURE 13: Lac repressor monomer has several domainsStructure from Protein Data Bank 1LBG. M. Lewis, et al., Science 271 (1996): 1247-1254. Photo courtesy of Hongli Zhan and Kathleen S. Matthews, Rice University.

lac Repressor Binding to the Operator Is Regulated by an Allosteric Change in Conformation

• Inducer binding causes a change in repressor conformation that reduces its affinity for DNA and releases it from the operator.

FIGURE 18: Inducer controls repressor conformation

lac Repressor Binding to the Operator Is Regulated by an Allosteric Change in Conformation

• Inducer binding causes a change in repressor conformation that reduces its affinity for DNA and releases it from the operator.

FIGURE 18: Inducer controls repressor conformation

RNA polymerase exists as a holoenzyme complex, complete with interacting transcriptional factors (TFs)

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RNA polymerase exists as a holoenzyme complex, complete with interacting transcriptional factors (TFs)

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RNA polymerase exists as a holoenzyme complex, complete with interacting transcriptional factors (TFs)

RNA polymerase exists as a holoenzyme complex, complete with interacting transcriptional factors (TFs) that can further interact with enhancer elements

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RNA polymerase exists as a holoenzyme complex, complete with interacting transcriptional factors (TFs) that can further interact with enhancer elements

RNA polymerase exists as a holoenzyme complex, complete with interacting transcriptional factors (TFs) that can further interact with enhancer elements… and even regulatory proteins that serve to interact with nucleosomes

Nuclear membrane

Modes of activation of inactive activators (TFs)

Other modifications can activate some transcription factors.

Slow process

Signaling pathways

“Receptor” in this case is a TF

“Receptor” in this case is a TF

“Receptor” in this case is a TF

Glucocorticord response element (GRE) can be in an enhancer

General types of interaction domains in activators (TFs)

• Helix-Loop-Helix motif facilitates protein-protein interaction. A separate basic region interacts with DNA.

• Leucine zipper motif facilitates protein-protein interaction. A separate basic region interacts with DNA.

• Homeodomain motif facilitates DNA binding • Zinc finger motif can facilitate DNA binding or protein

interaction

Protein-protein interaction facilitated by leucine zipper. The hydrophobic leucine region is on one side of an amphipathic helix. Basic regions bind DNA.

Protein-protein interaction facilitated by leucine zipper. The hydrophobic leucine region is on one side of an amphipathic helix. Basic regions bind DNA.

Protein-protein interaction facilitated by leucine zipper. The hydrophobic leucine region is on one side of an amphipathic helix. Basic regions bind DNA.

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Helix Loop Helix (HLH) proteins (~40-50 AAs)

Two amphipathic alpha helices (all charged residues on one side of the helix and all hydrophobic residues on other side) of 15-16 AAs in length separated by a loop that allows dimerization. The basic region interacts with DNA. Id can not interact with DNA but MyoD can.

Homodimers or Heterodimers

Only heterodimers shown

A A

Dimer binds two successive turns of the major groove of DNA. Binding of the ligand (hormone) to receptor increases its affinity

for DNA.

B

A B

Different spacing between the DNA binding sites leads to recruitment of different cofactors.

A

Different spacing between the DNA binding sites leads to recruitment of different cofactors.

A

A

Different spacing between the DNA binding sites leads to recruitment of different cofactors.

AB

General types of interaction domains in activators (TFs)

• Helix-Loop-Helix motif facilitates protein-protein interaction. A separate basic region interacts with DNA.

• Leucine zipper motif facilitates protein-protein interaction. A separate basic region interacts with DNA.

• Homeodomain motif facilitates DNA binding • Zinc finger motif can facilitate DNA binding or protein

interaction

• steroid receptor – Transcription factors that are activated by binding of a steroid ligand.

• helix-turn-helix – The motif that describes an arrangement of two α-helices that form a site that binds to DNA, one fitting into the major groove of DNA and the other lying across it.

• homeodomain – A DNA-binding motif that typifies a class of transcription factors.

The “homeodomain” forms a domain with three distinct helices

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Some proteins with a homeodomain function as repressors, some as activators.

Relative location of homeodomain differs in different “realizator” proteins

General types of interaction domains in activators (TFs)

• Helix-Loop-Helix motif facilitates protein-protein interaction. A separate basic region interacts with DNA.

• Homeodomain facilitates DNA binding • Leucine zipper motif facilitates protein-protein

interaction. A separate basic region interacts with DNA. • Zinc finger motif can facilitate DNA binding or protein

interaction

Usually more than one zinc finger (often 2-9) working together.

Protein-protein or protein-DNA interactions can be facilitated by zinc finger domains. Three helixes can fit into one turn of the major groove of DNA. Each alpha helix has at least two amino acids that make specific contacts with DNA. Non-conserved helix amino acids in different TF family members recognize different DNA sequences.

• Enhancers form complexes of activators that interact directly or indirectly with the promoter.

• Repressor Activity– proteins that inhibit expression of a gene. – in eukaryotes these generally act to prevent transcrip6on by

binding to an enhancer or silencer.

Regulation -Proximal and at a distance

• Methylation of a CpG island prevents activation of a promoter within it. • Repression is also caused by proteins that bind to

methylated CpG doublets… • Regulation and other methylated nucleic acids

• An enhancer activates the promoter nearest to itself, and can be any distance either upstream or downstream of the promoter.

• A UAS (upstream activating sequence) in yeast behaves like an enhancer, but works only upstream of the promoter… not so rigorous in other eukaryotes.

Enhancer action is independent of location

• Enhancers form complexes of activators that interact directly or indirectly with the promoter.

• Repressor Activity– proteins that inhibit expression of a gene. – in eukaryotes these generally act to prevent transcrip6on by

binding to an enhancer or silencer.

Regulation -Proximal and at a distance

• Methylation of a CpG island prevents activation of a promoter within it. • Repression is also caused by proteins that bind to

methylated CpG doublets… • Regulation and other methylated nucleic acids

DNA damage products formed after attack by oxygen radicals. dR = deoxyribose.Oxidatively damaged bases represent a third type of spontaneous lesion implicated in mutagenesis. Active oxygen species, such as superoxide radicals (O2·), hydrogen peroxide (H2O2), and hydroxyl radicals (OH·), are produced as by-products of normal aerobic metabolism.

Thymidine glycol, blocks DNA replication -if unrepaired, but has not yet been implicated in mutagenesis.

8-oxo-7-hydrodeoxyguanosine (8-oxodG, or GO) product (which frequently mispairs with A, results in a high level of G → T transversions).

DNA damage products formed after attack by oxygen radicals.

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Schematic presentation of the stimuli that induce MT and the downstream effects of MT overexpression. MT can be activated by a variety of stimuli, including metal ions, cytokines, growth factors, oxidative stress and radiation. Downstream effects of MT overexpression are modulation of transcription of both tumor suppressor protein p53 and nuclear transcription factor NF-κB. Another downstream effect of MT overexpression is free radical scavenging activity. All these downstream MT effects influence cell survival, cell growth, drug resistance and differentiation. Adopted and modified according to [107].

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Overview of metallothionein (MT) gene regulation and function. The MT promoter has MANY response elements that upregulate transcription. These include the following: (1) metal response elements (MRE), which are activated by the metal-responsive transcription factor (MTF-1) after zinc occupancy, which is a function of the dietary zinc supply; (2) glucocorticoid response elements (GRE); (3) elements activated by STAT (signal transducers and activators of transcription) proteins through cytokine signaling; and 4) the antioxidant (or electrophile) response element (ARE), activated in response to redox status.

Methylation may downregulate expression in some tumor cells.

Overview of metallothionein (MT) gene regulation and function. The MT promoter has MANY response elements that upregulate transcription. These include the following: (1) metal response elements (MRE), which are activated by the metal-responsive transcription factor (MTF-1) after zinc occupancy, which is a function of the dietary zinc supply; (2) glucocorticoid response elements (GRE); (3) elements activated by STAT (signal transducers and activators of transcription) proteins through cytokine signaling; and 4) the antioxidant (or electrophile) response element (ARE), activated in response to redox status.

Methylation may downregulate expression in some tumor cells.

Genes can be activated in multiple ways depending on which TF binding sites (BSs) are present in the promoter and which TFs are present in the cell. TFs that bind to response elements (DNA TFBSs) are activated by interaction with hormones or metals and/or other molecules that are only transiently present.

TFBSs

TFs

The metallothionein (MT) gene is normally expressed at a low level due to the constitutively expressed Transcriptional Factors, AP2 and AP1, binding to Basal Level Elements.

• Enhancers form complexes of activators that interact directly or indirectly with the promoter.

• Repressor Activity– proteins that inhibit expression of a gene. – in eukaryotes these generally act to prevent transcrip6on by

binding to an enhancer or silencer.

Regulation -Proximal and at a distance

• Methylation of a CpG island prevents activation of a promoter within it. • Repression is also caused by proteins that bind to

methylated CpG doublets… • Regulation and other methylated nucleic acids

• Demethylation at the 5′ end of many genes is often necessary for transcription to commence.

Restriction enzymes differ at methylated sites

Restriction digests can analyze methylation

• Demethylation at the 5′ end of many genes is often necessary for transcription to commence.

Restriction enzymes differ at methylated sites

Restriction digests can analyze methylation

• CpG islands often surround the promoters of constitutively expressed genes and where they are normally unmethylated.

• CpG islands are also found at the promoters of some tissue-regulated genes.

• There are ~29,000 CpG islands in the human genome.

CpG islands have clusters of CpG doublets:

Normally 1/100bps can increase to as many as 10/100bps

5’ CG 3’ | | GC

• Enhancers form complexes of activators that interact directly or indirectly with the promoter.

• Repressor Activity– proteins that inhibit expression of a gene. – in eukaryotes these generally act to prevent transcrip6on by

binding to an enhancer or silencer.

Regulation -Proximal and at a distance

• Methylation of a CpG island prevents activation of a promoter within it. • Repression is also caused by proteins that bind to

methylated CpG doublets… • Regulation and other methylated nucleic acids

• Enhancers form complexes of activators that interact directly or indirectly with the promoter.

• Repressor Activity– proteins that inhibit expression of a gene. – in eukaryotes these generally act to prevent transcrip6on by

binding to an enhancer or silencer.

Regulation -Proximal and at a distance

• Methylation of a CpG island prevents activation of a promoter within it. • Repression is also caused by proteins that bind to

methylated CpG doublets… • Regulation and other methylated nucleic acids

The 5′ End of Eukaryotic mRNA Is Capped

• The 5′ cap of most mRNA is monomethylated, but some small noncoding RNAs are trimethylated.

• The cap structure is recognized by protein factors to influence mRNA stability, splicing, export, and translation.

• The 5′ cap is formed by adding a G to the terminal base of the transcript via a 5′–5′ link.

• The capping process takes place during the transcription, which may be important for transcription reinitiation.

Eukaryotic mRNA has a methylated 5’ cap

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5’ capping Helps to Overcome Transcriptional pausing

• DNA-directed RNA polymerase II subunit RPB1 - an enzyme that in humans is encoded by the POLR2A gene. RPB1 is the largest subunit of RNA polymerase II. It contains a carboxy terminal domain (CTD) composed of up to 52 heptapeptide repeats (YSPTSPS)

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• DNA-directed RNA polymerase II subunit RPB1 - an enzyme that in humans is encoded by the POLR2A gene. RPB1 is the largest subunit of RNA polymerase II. It contains a carboxy terminal domain (CTD) composed of up to 52 heptapeptide repeats (YSPTSPS)

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• DNA-directed RNA polymerase II subunit RPB1 - an enzyme that in humans is encoded by the POLR2A gene. RPB1 is the largest subunit of RNA polymerase II. It contains a carboxy terminal domain (CTD) composed of up to 52 heptapeptide repeats (YSPTSPS)

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Eukaryotic mRNA is modified, processed, and transported

Splicing is required for mRNA export

Splicing is required for mRNA export

Splicing is required for mRNA export

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Splicing is required for mRNA export

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Splicing is required for mRNA export

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Splicing is required for mRNA export

Thus, Splicing Can Be Temporally and Functionally Coupled with Multiple Steps in Gene Expression

• Splicing in the nucleus can influence mRNA translation in the cytoplasm.

• nonsense-mediated mRNA decay (NMD) – A pathway that degrades an mRNA that has a nonsense mutation prior to the last exon.

The EJC complex couples splicing with NMD

Eukaryotic mRNA is modified, processed, and transported

Nuclear Splice Junctions Are Short Sequences

• There exist minor introns relative to the major introns that follow the GU-AG rule.

• Minor introns follow a for more general AU-AC rule with a different set of consensus sequences at the exon–intron boundaries.

• There exist minor introns relative to the major introns that follow the GU-AG rule.

• Minor introns follow a for more general AU-AC rule with a different set of consensus sequences at the exon–intron boundaries.

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8346

8446

Correct splicing removes three introns by pairwise recognition of the junctions

Pre-mRNA Splicing Proceeds through a Lariat

• The intron is released as a lariat when it is cleaved at the 3′ splice site, and the left and right exons are then ligated together.

Splicing proceeds through formation of a lariat

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Splicing uses simple basic and simple “transester-ification“ reactions

Splicing uses simple basic and simple “trans -esterification“ reactions

Splicing uses simple basic and simple “transesterification”reactions

Splicing uses simple basic and simple “transesterification” reactions