Transcription in Eukaryotes Practice Flashcards

Learning Objectives for Eukaryotic Transcription

• Match molecular processes or the flow of information (Central Dogma of Molecular Biology) to specific locations within a eukaryotic cell.

• Identify the different parts of a eukaryotic gene and describe their distinct roles in biological processes.

• Describe and order the steps involved in the recognition, initiation, elongation, and termination of transcription in a eukaryotic cell.

• Describe the roles of specific DNA regions and enzymes involved in the transcription process in eukaryotes.

• Compare and contrast the transcription process in bacteria versus eukaryotes, highlighting differences in gene structure and components like promoters.

Fundamental Rules and Characteristics of Eukaryotic Transcription

• Transcription Unit Definition: Eukaryotic protein-coding genes are typically monocistronic, meaning only one region is translated into a single protein from the resulting mRNA.

• Gene Structure: Eukaryotic genes consist of a single coding region flanked by Untranslated Regions (UTRs). This coding region is discontinuous, being broken up into exons (expressed sequences) and introns (intervening sequences).

• Spatial and Temporal Context:

  • Location: Transcription occurs exclusively in the nucleus.

  • Timing: It occurs before translation and usually happens during the $G_0$ (nondividing) phase, or the $G_1$ and $G_2$ phases when genes coding for cellular organelle proteins are synthesized.

• Selectivity of Transcription:

  • Start Site: Transcription begins at the $+1$ site, which is the first base of Exon 1 and part of the $5'$ UTR.

  • End Site: Transcription ends in the termination region located within the $3'$ UTR.

  • Clarification: Transcription does NOT start at the $AUG$ start codon; that is the starting point for translation.

• Transcribed Components: Both exons and introns are transcribed into the initial RNA molecule. The $5'$ UTR and $3'$ UTR may be part or all of the first and last exons, respectively, and are also transcribed into the pre-mRNA.

• Synthesis Directionality:

  • RNA is transcribed from single-stranded DNA ($ssDNA$) templates.

  • RNA molecules are antiparallel and complementary to the DNA template strand.

  • Synthesis always proceeds in the $5' \rightarrow 3'$ direction relative to the growing RNA molecule, with nucleotides added to the $3'$ end.

  • Only one strand is transcribed per gene, though the template strand can vary between different genes.

Eukaryotic Promoter Structure and Consensus Sequences

• Promoter Complexity: Eukaryotic promoters are significantly more complex than prokaryotic ones. They contain short sequences known as motifs or consensus sequences critical for recruiting RNA Polymerase to the DNA.

• Core Promoter:

  • Acts as the primary "ON/OFF" switch for the gene.

  • It is a highly conserved sequence serving as the binding site for General Transcription Factors (GTFs) and RNA Polymerase II.

  • It contains the TATA box ($TATAAA$ motif), which is a signature sequence recognized by the TATA Binding Protein (TBP).

  • Found in approximately $25\%$ of human promoters; only $10\%$ contain the exact $TATAAA$ sequence, yet TBP is required for transcription even when the motif is absent.

• Proximal Promoter:

  • Located near the core promoter and contains regulatory sequences.

  • Binds Specific Transcription Factors (activator proteins) that stimulate and stabilize the activity of RNA Polymerase II at the core promoter.

• Additional Elements: Other functional sequences not always shown in simplified diagrams include the Downstream Promoter Element (DPE) and the TFIIB Recognition Element (BRE).

• Distal Regulatory Regions: Enhancers and silencers can be located thousands of base pairs away from the transcription start site, influencing initiation through DNA looping.

Key Proteins and Molecular Complexes

• Transcription Factors (TFs): Proteins or complexes that bind to promoter or regulatory sequences to influence transcription initiation through interactions with RNA polymerase.

  • General TFs: Involved in the transcription of most genes (e.g., TBP).

  • Specific TFs: Regulatory proteins that differ between genes (e.g., $SRY$ in humans).

• TATA Binding Protein (TBP):

  • A component of the TFIID complex.

  • Attaches by interacting with negatively charged phosphates in the DNA backbone.

  • Inserts amino acid side chains between bases, stabilized by hydrogen bonds.

  • Distorts and bends the DNA to "melt" it (breaking hydrogen bonds between base pairs).

• Mediator Complex: Acts as a bridge between Specific TFs bound to distal regulatory regions (enhancers) and the RNA Polymerase II holoenzyme bound at the core promoter.

• Eukaryotic RNA Polymerases:

  • RNA Polymerase I: Transcribes large rRNAs.

  • RNA Polymerase II: Transcribes pre-mRNA, some snRNAs, snoRNAs, and some miRNAs. It consists of almost 60 subunits.

  • RNA Polymerase III: Transcribes tRNAs, small rRNAs, some snRNAs, and some miRNAs.

  • RNA Polymerase IV: Found in plants; transcribes some siRNAs.

The Stages of Eukaryotic Transcription

1. Recognition

• TFIID complex, containing TBP, recognizes and binds to the TATA box in the core promoter.

• Binding of TFIID recruits other General Transcription Factors required for RNA Polymerase II ($RNAPII$) recognition and initiation.

2. Initiation

• Minimal Initiation Complex (MIC): TFIIB recruits $RNAPII$ and other GTFs.

• Pre-Initiation Complex (PIC): Formed by the binding of $RNAPII$ complexed with GTFs (like TFIIH) and the mediator complex. This creates the RNA Polymerase II Holoenzyme.

• Activation of $RNAPII$:

  • TFIIH Functions: Acts as a helicase to open the transcription bubble and as a kinase to phosphorylate the CTD Tail (Carboxyl-terminal Domain) of $RNAPII$.

  • Phosphorylation of the CTD tail transitions the complex into an active state.

• Release: The polymerase clears the promoter after transcribing approximately 30 bases. Some GTFs (TFIIB, TFIIA) are released, while TFIID remains at the promoter to facilitate subsequent rounds of recognition.

3. Elongation

• Elongation Factors (EFs): Required for high processivity (the stable synthesis of long nucleic acids).

• EF Functions:

  • Evict histones ahead of $RNAPII$ and replace them behind the moving bubble (managing Nucleosomes/Chromatosomes).

  • Maintain the heteroduplex RNA:DNA hybrid region.

  • Navigate and resolve issues like DNA lesions or problematic sequences that might stall transcription.

• $RNAPII$ maintains a transcription bubble, opening the DNA ahead and closing it behind the complex.

4. Termination

• Transcription stops when specific termination sequences are transcribed.

• This leads to the cleavage of the pre-mRNA transcript at a specific position in the $3'$ UTR.

• Linked to the de-phosphorylation (deactivation) of the $RNAPII$ CTD tail, causing the enzyme to drop off the DNA template.

Comparative Analysis: Eukaryotes vs. Bacteria

• Comparison of Insertion Scenarios: If a full-length eukaryotic gene is inserted into a bacterial chromosome, it often fails to produce functional protein due to:

  1. Bacterial RNA Polymerase and sigma factors do not recognize eukaryotic promoter sequences.

  2. Bacteria lack the machinery to remove introns (splicing).

  3. Missing bacterial-specific promoter regions ($-10$, $-35$).

  4. Missing bacterial-specific termination sequences.

  5. Missing bacterial translation ribosome binding sites (Shine-Dalgarno sequence).

• Recommended Changes for Expression in Bacteria:

  1. Use a cDNA copy of the gene (coding sequences/exons only).

  2. Insert a strong bacterial promoter upstream.

  3. Add an intrinsic termination sequence or $rut$ site downstream/upstream.

  4. Add a bacterial translation initiation sequence.

Questions & Discussion

• What if TBP was inhibited from binding at the promoter?: This would cause an immediate halt to transcription recognition and initiation, as TBP is an essential component of the TFIID complex required to recruit RNA Pol II to the core promoter.

• How does the CTD tail regulate the process?: Adding phosphate groups (via TFIIH) activates the polymerase for initiation and elongation, while removing phosphate groups is a signal for termination and deactivation.