Transcription and Post-Transcriptional RNA Processing in Eukaryotes and Prokaryotes

Transcription: Overview and Mechanism

Definition and Process

Transcription is the process by which DNA is used as a template to synthesize RNA. This is a fundamental step in gene expression, allowing genetic information to be transferred from DNA to RNA, which can then be translated into proteins.

• RNA polymerase is the enzyme responsible for transcribing DNA into RNA.

• There are multiple types of RNA polymerases, each with specific roles.

• RNA transcripts are synthesized in the 5' to 3' direction by adding nucleoside triphosphates (NTPs) onto the 3' OH group.

• Each gene is transcribed using only one strand of the double-stranded DNA (the template strand).

• Upstream refers to regions of DNA before the gene start site; downstream refers to regions after the gene start site.

• Transcription occurs in three stages: initiation, elongation, and termination. These stages differ slightly between prokaryotes and eukaryotes.

Example: The coding strand and template strand are shown, with RNA polymerase synthesizing the RNA strand complementary to the template strand.

Prokaryotic Transcription

Initiation

Prokaryotic transcription initiation requires specific DNA sequences and protein factors.

• Promoters are DNA sequences that signal the start of transcription.

• Promoter consensus sequences include:

  • Pribnow box: Located ~10 bp upstream of the start site (sequence: TATAAT).

  • -35 base pair consensus sequence.

  • Occasionally, a -40 to -60 upstream sequence.

• The RNA polymerase holoenzyme binds to the promoter, consisting of a sigma factor and a core enzyme.

• The sigma factor is a peptide sequence that controls specificity of RNA polymerase binding.

Example: Diagram showing promoter, sigma factor, RNA polymerase, and DNA helix during initiation.

Elongation and Termination

• Elongation occurs after initiation, with the formation of a transcription bubble (~18 nucleotides of unwound DNA).

• Termination occurs when RNA polymerase reaches a specific sequence:

  • Termination sequences (terminators) are found upstream of the termination site.

  • Rho-dependent terminators require the rho protein for termination.

  • Rho-independent terminators function without the rho protein, often in uracil-rich RNA regions.

  • Intrinsic termination occurs in uracil-rich RNA transcripts due to weak bonds.

Example: Diagram showing initiation, elongation, and termination, with a single terminator at the end of a group of genes (polycistronic RNA).

Eukaryotic Transcription

RNA Polymerases and Initiation

Eukaryotic transcription is more complex than prokaryotic transcription, involving multiple RNA polymerases and regulatory factors.

• Diverse RNA polymerases transcribe different RNAs:

RNA Polymerase	Transcribes
RNA polymerase I	Ribosomal RNA (rRNA)
RNA polymerase II	Messenger RNA (mRNA)
RNA polymerase III	Transfer RNA (tRNA)

• Initiation requires general transcription factors (GTFs) such as TFIIA, TFIIB, TFIID.

• Promoter regions include the TATA-box (~30 bp upstream of start site), which binds TFIID (TATA binding protein) and recruits GTFs.

• The preinitiation complex includes GTFs and RNA polymerase II at the promoter.

Example: Diagram showing RNAP, GTFs, and DNA at the promoter region.

Elongation and Termination

• The carboxyl terminal domain (CTD) of RNA polymerase II controls elongation.

• Phosphorylation of the CTD by GTFs triggers release of RNA polymerase II from the initiation complex and allows elongation.

• RNA polymerase II transcribes hundreds to thousands of nucleotides past the coding sequence; RNA processing forms the mature mRNA.

Example: Diagram showing RNAP elongating the transcript.

Transcriptional Regulation

• Enhancers activate and enhance transcription.

• Silencers repress transcription.

• Specific transcription factors activate or repress specific genes.

• These factors can be located near or far from the transcription start site.

• Cis-acting elements are found within the same chromosome as the gene they regulate.

Post-Transcriptional RNA Processing

RNA Processing Steps

After transcription, RNA undergoes several processing steps before translation.

• A 5' cap is added via attachment of a 7-methylguanosine molecule, protecting RNA from degradation and aiding translation.

• A 3' polyadenylation tail is added by incorporating 150-200 adenine nucleotides at the end, triggered by a polyadenylation signal (AAUAAA).

Example: Diagram showing protein coding mRNA with 5' cap, coding sequence, and poly-A tail.

RNA Splicing

• Splicing removes non-coding introns from the pre-mRNA, joining exons.

• The spliceosome is a complex of small nuclear RNAs (snRNAs: U1, U2, U4, U5, U6) and proteins, forming the small ribonucleoprotein complex (snRNP).

• Splice sites:

  • 5' splice site: GU

  • 3' splice site: AG (GU-AG rule)

Example: Diagram showing removal of introns and joining of exons in mRNA splicing.

RNA Editing

• RNA editing is a post-transcriptional modification that alters nucleotide sequences.

• Substitution editing: nucleotide is changed.

• Insertional editing: nucleotide is added.

• Deletion editing: nucleotide is deleted.

• Guide RNAs determine where editing occurs.

Example: Diagram showing guide RNA directing editing complex.

RNA Interference (RNAi)

Mechanism and Types

RNA interference is a form of post-transcriptional regulation that silences gene expression through RNA molecules.

• miRNAs (microRNAs): single-stranded RNAs that target degradation of many RNA transcripts.

• miRNAs are transcribed as part of longer RNAs, processed by Dicer into ~22 nucleotide miRNAs.

• RISC complex binds miRNA, targeting specific transcripts for degradation.

• siRNAs (small interfering RNAs): double-stranded RNAs that target degradation of specific transcripts.

• siRNAs are processed by Dicer and loaded into RISC, which unwinds and binds the target RNA for degradation.

Example: Diagram showing Dicer processing and RISC targeting RNA for degradation.

Practice Questions

1. Which of the following polymerases is responsible for transcribing mRNA in eukaryotes?

   • a. RNA polymerase I

   • b. RNA polymerase II

   • c. RNA polymerase III

   • d. RNA polymerase

2. Which of the following general transcription factors is required for transcription initiation?

   • a. TFIIA

   • b. TFIIB

   • c. TFIID

   • d. TFIIH

3. Which of the following modifications occurs to the RNA polymerase tail in order to trigger it to elongate the transcript?

   • a. Methylation

   • b. Acetylation

   • c. Carboxylation

   • d. Phosphorylation

4. Which of the following is a specific transcription factor?

   • a. Silencers

   • b. Specific transcription factors

   • c. Enhancers

   • d. Promoters

5. True or False: siRNAs target a variety of different RNA transcripts for degradation.

   • a. True

   • b. False

6. Which of the following enzymes are responsible for cleaving and processing miRNAs and siRNAs?

   • a. DICER

   • b. RISC

   • c. Guide RNA enzymes

   • d. RNA polymerase II

7. Which of the following is responsible for splicing introns out of the pre-mRNA?

   • a. RNA polymerase III

   • b. The gene promoter

   • c. A gene transcript

   • d. The spliceosome

Key Equations and Concepts

• Direction of RNA synthesis: $5' \rightarrow 3'$

• Consensus sequence (Pribnow box): $\text{TATAAT}$

• Polyadenylation signal: $\text{AAUAAA}$

Additional info: Some explanations and terminology have been expanded for clarity and completeness, including the roles of transcription factors, RNA processing steps, and the mechanisms of RNA interference.