Eukaryotic Transcription and RNA Processing

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Chapter 12: Eukaryotic Transcription and RNA Editing

Introduction

This chapter explores the mechanisms of eukaryotic transcription and the post-transcriptional modifications that are essential for the maturation of RNA. Key processes include transcription initiation, elongation, termination, 5’ capping, polyadenylation, RNA splicing, and RNA editing. Understanding these processes is fundamental for comprehending gene expression regulation in eukaryotes.

Eukaryotic RNA Polymerases

Types and Functions

Eukaryotic cells possess three main nuclear RNA polymerases, each responsible for transcribing different classes of genes:

RNA Polymerase I: Transcribes all rRNA genes except 5S rRNA.
RNA Polymerase II: Transcribes all protein-coding genes (mRNAs) and some snRNA genes required for splicing.
RNA Polymerase III: Transcribes all tRNA genes, the 5S rRNA gene, and microRNA genes.

Each polymerase recognizes distinct promoter elements and requires specific transcription factors for initiation.

Transcription Initiation in Eukaryotes

Promoter Structure

The promoter is a DNA sequence upstream of the gene that serves as the binding site for transcription machinery. Key elements include the TATA box, CAAT box, and GC-rich regions.

TATA Box: Located ~30 bp upstream of the transcription start site; recognized by TATA-binding protein (TBP).
CAAT Box and GC Box: Promoter-proximal elements that enhance transcription efficiency.

Diagram of eukaryotic promoter elements including TATA box, CAAT box, and GC-rich box

Assembly of the Pre-Initiation Complex

Transcription initiation involves the sequential binding of general transcription factors (GTFs) and RNA polymerase II to the promoter:

TFIID (containing TBP) binds the TATA box.
Other GTFs (TFIIA, TFIIB, TFIIE, TFIIF, TFIIH) assemble, forming the pre-initiation complex.
RNA polymerase II is recruited and positioned at the transcription start site.

Stepwise assembly of transcription factors and RNA polymerase II at the promoter

Initiation and Promoter Escape

After the pre-initiation complex forms, RNA polymerase II synthesizes short RNA transcripts. TFIIB can cause abortive initiation, where short transcripts are released until the polymerase escapes the promoter and enters productive elongation.

Stages of transcription initiation and promoter escape

Transcription Elongation

Process and Regulation

During elongation, RNA polymerase II moves along the DNA, synthesizing RNA in the 5’ to 3’ direction. Elongation is regulated by pausing and the action of elongation factors.

Elongation Pausing: RNA polymerase may pause shortly after initiation, regulated by factors such as NELF and DSIF.
Elongation Factors: P-TEFb phosphorylates components of the paused complex, allowing productive elongation.

Diagram showing promoter-proximal pausing and release during elongation

Transcription Termination and Polyadenylation

Termination Mechanism

Termination of transcription by RNA polymerase II involves recognition of the polyadenylation signal (AAUAAA) in the nascent RNA. Several protein factors are involved:

CFI and CFII: Cleavage factors that cut the RNA downstream of the polyadenylation signal.
CPSF: Cleavage and Polyadenylation Specificity Factor, binds the polyadenylation signal.
CstF: Cleavage Stimulation Factor, interacts with CPSF to promote cleavage.
PAP: Poly(A) polymerase adds a poly(A) tail to the 3’ end of the RNA.

Steps in transcription termination and polyadenylation

5’ Capping of mRNA

Mechanism and Purpose

Shortly after transcription initiation, a 7-methylguanosine cap is added to the 5’ end of the nascent mRNA. This process involves:

Removal of the terminal phosphate from the 5’ triphosphate end by RNA 5’-triphosphatase.
Addition of GMP by guanylyltransferase.
Methylation of the guanine base by methyltransferase.

The 5’ cap protects mRNA from degradation, facilitates ribosome binding during translation, and is involved in nuclear export.

Attachment of 7-methylguanosine cap to the 5’ end of mRNA

RNA Splicing

Removal of Introns

RNA splicing is the process by which non-coding introns are removed from pre-mRNA and exons are joined to form mature mRNA. The spliceosome, a complex of snRNAs and proteins, catalyzes this process.

Splice Sites: Conserved sequences at the exon-intron boundaries guide the spliceosome.
Lariat Formation: The intron is excised as a lariat structure and degraded.

Diagram of RNA splicing showing intron removal and exon ligation

RNA Editing

Post-Transcriptional Base Modification

RNA editing refers to changes in the nucleotide sequence of an RNA molecule after it has been synthesized. This can involve:

Addition or deletion of specific bases.
Chemical modification of bases, such as deamination (e.g., cytosine to uracil, adenosine to inosine).

RNA editing increases transcriptome diversity and can affect protein function.

Chemical reactions involved in RNA editing, such as cytidine deamination

Summary Table: Eukaryotic RNA Polymerases

Polymerase	Transcribes
RNA pol I	rRNA genes (except 5S rRNA)
RNA pol II	Protein-coding genes (mRNA), some snRNA genes
RNA pol III	tRNA genes, 5S rRNA gene, microRNA genes

Key Terms

Promoter: DNA sequence where transcription machinery assembles.
Transcription Factors: Proteins required for the initiation of transcription.
Spliceosome: Complex responsible for removing introns from pre-mRNA.
Polyadenylation: Addition of a poly(A) tail to the 3’ end of mRNA.
RNA Editing: Post-transcriptional modification of RNA sequence.