Transcription Regulation in Eukaryotes

Eukaryotic Gene Expression: Transcription

• Eukaryotic transcription takes place on a densely proteinized chromatin template.
• Bacterial RNA polymerase reads DNA; eukaryotic RNA polymerase cannot directly read DNA.
• Transcription requires sequence-specific DNA-binding transcription factors (TFs).
• Core RNA polymerase II machinery is essential.
• Coregulators bridge TFs to the transcriptional machinery.
• Chromatin remodeling factors mobilize nucleosomes.
• Enzymes catalyze covalent modifications of histones and other proteins.
• Eukaryotic transcription is usually under positive regulation.
• Opening chromatin and binding TFs to the binding sites precede polymerase binding.
• Polymerase binds to promoter and TFs bind to enhancers.

Eukaryotic Transcriptional Regulation

• Eukaryotic genomes fold into topologically associated domains (TADs).
• Chromosomes with low gene density reside at the nuclear periphery.
• Chromosomes with high gene density occupy the nuclear interior.
• Transcriptionally active genes associate with nuclear pore complexes.
• Transcription decondenses chromosome territories, loops back into condensed territories when transcription ceases.
• The banding pattern of polytene chromosomes corresponds to TADs.

Nuclear Architecture and Premature Aging Syndromes

• A link exists between disrupted nuclear membrane and premature aging syndromes.
• Nuclear lamina: a protein meshwork underlying the nuclear membrane composed of intermediate filaments.
• Nuclear lamina provides mechanical support, regulates cellular events (DNA replication), and participates in chromatin organization.
• It anchors nuclear pore complexes and is a platform for protein complexes involved in signal transduction pathways.

Eukaryotic Gene Expression: Transcription

• Some eukaryotic TFs function by introducing DNA bends, facilitating interactions of other components.

Nucleic Acid Recognition by Proteins

• DNA-binding proteins incorporate domains that facilitate the binding of nucleic acid.
• These proteins interact with DNA ends (e.g., DNA ligases, exonucleases), enclose DNA, bind to the face of DNA's helix, and possess motifs that fit into the major grove of DNA for higher stability.

Long Range Regulatory Elements

• Enhancers: typical mammalian genomes contain many enhancers (1-2% contribute to gene regulation); ranging from 100-500 bp in length; can increase or decrease the promoter.
• Silencers: decrease gene promotor activity; between 700 and 1000 bp.

Initiation in Eukaryotic Promoters

• Various basal transcription factors (BTFs) bind to cis-acting elements to form the initiation complex for RNA polymerase.
• Basal factors are identified as TFIIA, TFB, TFIIIC, TFIIID, etc.
• Core promoter motifs for RNA polymerase II are present.

Eukaryotic RNA Polymerases and Promoter Recognition

• Eukaryotes have multiple nuclear DNA-dependent RNA polymerases (I, II, III).
• Different RNA polymerases transcribe different products within the cell compartment (e.g., rRNA, mRNA, tRNA).

RNA Polymerases II Promoters

• Enhancers and silencers increase or decrease the expression of a gene.
• Enhancers function upstream (or downstream) of the promoter.
• Enhancers and silencers may be separated from the promoter by distances as large as 100 kb.

Enhancers Assist Initiation

• Enhancers activate an accessible promoter located virtually anywhere from the promoter either upstream or downstream.
• Enhancers increase activator concentration.

How Enhancers Work

• Enhancers normally work in cis configuration, with a target promoter; enhancers can be made to work in trans configuration;
• The principle is that enhancers work in any situation and distance from the promoter.
• Enhancers increase activator concentration in the vicinity of the promoter from cis.

Role of Coactivators and Corepressors

• Coactivators increase or corepressors decrease transcriptional activity.
• Coactivators can be chromatin modification complexes (histone modifications) or chromatin remodeling complexes.

Eukaryotic RNA Polymerases

• Typical eukaryotic RNA polymerases have 12 subunits (2 large, many smaller).
• RNA polymerase II is the most characterized.
• The largest subunit in RNA polymerase II has a carboxy-terminal domain (CTD).

Phosphorylation of the RNA Polymerase II C-Terminal Doamin (CTD)

• CTD has a repeating sequence of seven residues (Y1S2P3T4S5P6S7); the number of repeats can differ among species.
• Phosphorylation status affects mRNA processing stage transitions

Mitochondrial and Chloroplast RNA Polymerases

• Mitochondrial and chloroplast RNA polymerases are smaller than bacterial enzymes.
• Eukaryotic genomes have fewer genes to transcribe and simpler regulation.

RNA Polymerase I

• RNA polymerase I directs RNA synthesis in the nucleoli and transcribes ribosomal RNA.

RNA Polymerase III Promoter Organization

• RNA polymerase III uses two classes of promoters: internal promoters and external promoters.
• Specific promoter elements and their positioning are different for internal versus external promoters.

Interactions at Internal Promoters

• Protein-protein interactions assist in the assembly of polymerase with basal factors.
• TAFs and TFB bind to different regions of the promoter.

Interactions at External Promotors

• TATA boxes, proximal sequence elements (PSEs), and octamer elements aid in basal transcription and efficiency.

Chromatin Remodeling Exposes the Promoter

• Chromatin remodeling occurs before the initiation of transcription, as nucleosomes must be moved away from the promoter to expose it to transcription factors.

TATA Binding Proteins (TBP): A Nearly Universal Factor

• TBP is a component of the positioning factor (interaction with RNA polymerase I/II/III) required for promoter binding.

TATA-Less Pre-initiation Complex

• TATA-less promoters rely on other elements for positioning and can be found in housekeeping and developmental genes.
• TBP has similar positioning in TATA-less promoters.

Gene Promoters and Chromatin Categories

• The inaccessibility of DNA due to histone proteins correlates to various chromatin categories.
• Poised genes require secondary signals while active genes only require basal TFs.

Transcription Basal Apparatus Assembles at the Promoter

• TFIID is the first general TF to associate with template DNA.
• Factors such as activators, UPEs and UPEs' binding factors increase the frequency of initiation.
• Binding TFIID to TATA box or Inr triggers the first step of initiation in transcription.

Transcription Basal Apparatus Assembles at the Promoter

• TFIIH is a complex TF and is involved in DNA repair.
• TFIIH phosphorylates CTD tail of RNA pol II, which is involved in release for further progress to elongation.

Mediator: A Molecular Drawbridge

• Mediator connects transcriptional activators (bound at enhancers and other regulatory elements) to RNA polymerase II
• The mediator links the initiation complex with distal elements by acting as a drawbridge.

Initiation of Transcription

• Multistage process: includes promoter melting, abortive initiation (short transcripts), promoter escape, pausing, and pause release.

Promoter Clearance and Elongation

• DNA strands separate following RNA pol binding.
• Promoter clearance is a crucial determinant for poised or active gene transcription.
• Enhancers influence the process.

The C-terminal Domain (CTD) of RNA Pol II

• CTD is required for capping and splicing; a repetitive structure emerged via evolution.
• CTD contains repeats (7 amino acids), linking transcriptional and RNA processing.
• Phosphorylation of Serines in the CTD is crucial for releasing the RNA Pol II for elongation.

Co-transcriptional Processing

• Transcription and co-transcriptional processing are tightly linked and performed via multifunctional proteins (e.g., capping enzyme complex (CEC)).
• The processing occurs concurrently with transcription, so RNA processing begins immediately after transcription, using 5' capping as an initial example.

The Cap-Binding Complex

• Cap binds to the cap-binding complex (CBC), which is identified by the nuclear pore.
• This complex facilitates mRNA export and prevents decapping enzymes.
• CBC is exchanged with other factors during mRNA transport and translation.

Nuclear Import and Export

• Small and large molecules are actively transported across the nuclear membrane.
• Importins and exportins (karyopherins) are the receptors involved in this process, and Ran is a vital factor for these processes.

The Ran Cycle

• Ran is a critical factor for nuclear transport and regulates processes within the import/export of molecules into and out of the nucleus.

Regulated Nuclear Import and Export of TFs and Proteins

• Regulatory proteins need to be directed to their corresponding nuclear activity sites.
• The receptors are known as importins and exportins (karyopherins) and mediate nuclear import and export pathways.

Nuclear Import and Export: TFs, Proteins

• Mechanisms in nuclear import/export: importin/Ran-dependent processes and nuclear import, and nuclear export.

Termination of Transcription

• The process is initiated by recognition of the sequence AAUAAA in Poly(A) sites.
• The endonuclease cleaves RNA, and Poly(A) polymerase adds 200 A residues to the 3' end.

Cleavage and Poly(A) at the 3' end

• CPSF/CstF and PAP proteins are part of the 3' processing complex for mRNA cleavage and polyadenylation as well as degradation avoidance.

Histone mRNA 3ʹ End Formation

• Histone mRNAs lack introns and contain HDE sequences at their 3ʹ ends, which are recognized by factors and regulate their localization.
• These elements are vital for mRNA regulation.

Summary: Initiation is Followed by Promoter Clearance and Elongation

• RNA pol II promoters' key regulated steps are promoter clearance and elongation, controlled via enhancers and phosphorylation of the CTD tail.
• These events involve TFs and accessory factors.

Other Core Promotor Elements

• Additional elements (e.g, BRE, Inr, MTE, DPE) help regulate transcription initiation in promoters lacking TATA box; TATA box, and upstream promoter elements (UPEs) are present.

Transcription Elongation Through the Nucleosome Barrier

• Nucleosomes serve as barriers to elongating RNA polymerases II.
• Chromatin remodeling complexes (e.g., FACT, elongation factors, and TFIIS) facilitate the movement of the RNA polymerase over nucleosomes.

Co-transcriptional Processing Revisited

• Transcription and processing are coupled, utilizing various factors to ensure timely and accurate transcript production and modification (e.g., capping, splicing).