Premed · Premed · Biochemistry
Lecture 26: Transcription and RNA Processing
Biochemistry
Learning Objectives
By the end of this lecture, students will be able to:
- Describe the mechanism of transcription by RNA polymerase in prokaryotes and eukaryotes
- Identify the promoter elements and transcription factors required for eukaryotic gene expression
- Explain the three major co-transcriptional and post-transcriptional RNA processing events in eukaryotes
- Describe the mechanism and significance of RNA splicing, including the role of snRNPs and the spliceosome
- Explain the regulation of gene expression at the transcriptional level
- Discuss clinical disorders caused by defects in transcription and RNA processing
Lecture Content
I. Overview of Transcription
Transcription is the process by which RNA is synthesized from a DNA template by RNA polymerase. During this process, RNA polymerase reads the template strand (also called the antisense or minus strand) in the 3' to 5' direction. The other strand, known as the coding strand (sense or plus strand), has the same sequence as the RNA transcript, except that thymine replaces uracil. Like DNA synthesis, RNA is synthesized in the 5' to 3' direction, and the substrates are NTPs (ATP, GTP, CTP, and UTP), with pyrophosphate (PPi) released during each nucleotide addition.
A key distinction between transcription and DNA replication is that RNA polymerase does NOT require a primer to begin synthesis, unlike DNA polymerase. Furthermore, transcription operates with lower fidelity than DNA replication, making approximately one error per 10^4 to 10^5 nucleotides without a proofreading exonuclease. This lower fidelity is acceptable because RNA molecules are transient and cells produce multiple copies of each transcript.
II. Prokaryotic Transcription
In prokaryotes, a single RNA polymerase (with the core enzyme composition alpha2-beta-beta'-omega) transcribes all types of RNA. The sigma factor (sigma-70 being the housekeeping factor) associates with the core enzyme to form the holoenzyme. The sigma factor is responsible for recognizing the promoter and enabling specific initiation. After approximately 10 nucleotides of RNA have been synthesized, sigma dissociates from the complex in a step known as promoter clearance.
Promoter Elements (prokaryotic):
Prokaryotic promoters contain two critical consensus elements. The -10 region (Pribnow box) has the consensus sequence TATAAT and is AT-rich to facilitate easy melting of the DNA. The -35 region has the consensus sequence TTGACA and is recognized directly by the sigma factor. The spacing between these two elements is critical, typically around 17 base pairs.
Stages:
Prokaryotic transcription proceeds through three stages. During initiation, the holoenzyme binds the promoter to form a closed complex, followed by melting of approximately 14 bp of DNA to form the open complex, and then the first phosphodiester bond is formed without a primer. During elongation, sigma dissociates and the core enzyme moves along the template, synthesizing RNA at approximately 40-80 nucleotides per second while the transcription bubble moves with the polymerase. Termination occurs by one of two mechanisms. In rho-independent termination, a GC-rich palindrome forms a hairpin in the nascent RNA followed by a run of U residues that form weak rU-dA base pairs, causing the RNA to dissociate from the template. In rho-dependent termination, the Rho protein, which functions as a helicase, translocates along the RNA and unwinds the RNA-DNA hybrid at a pause site.
Antibiotics Targeting Prokaryotic Transcription:
Two important antibiotics target prokaryotic transcription. Rifampin binds the beta-subunit of bacterial RNA polymerase and blocks the initiation of RNA synthesis. It is used clinically to treat tuberculosis and as prophylaxis for meningococcal meningitis. Actinomycin D intercalates into DNA and blocks elongation; it is used experimentally and as an anticancer agent.
III. Eukaryotic Transcription
Eukaryotic cells possess three nuclear RNA polymerases, each transcribing different classes of RNA. RNA Pol I transcribes ribosomal RNA (28S, 18S, and 5.8S) in the nucleolus and is the most active polymerase, producing approximately 80% of total RNA. RNA Pol II transcribes messenger RNA as well as most snRNAs and miRNAs, and is the target of most transcriptional regulation. RNA Pol III transcribes transfer RNA, 5S rRNA, and other small RNAs. In addition, mitochondria have their own RNA polymerase, which is a single-subunit enzyme similar to bacteriophage T7 RNA polymerase. A clinically important toxin, alpha-amanitin from the Amanita phalloides mushroom, is a potent inhibitor of RNA Pol II (and Pol III at higher doses), and its ingestion causes liver failure.
Eukaryotic Promoter Elements (for RNA Pol II):
The core promoter contains the TATA box, located approximately 25-30 bp upstream of the transcription start site (+1), with the consensus sequence TATAAA. The TATA box is bound by TBP (TATA-binding protein), a subunit of TFIID. Not all promoters contain a TATA box; some instead use an Inr (initiator) element or a DPE (downstream promoter element).
Proximal promoter elements, located approximately 50-200 bp upstream, include the CAAT box and GC box. These are bound by specific transcription factors such as Sp1, which recognizes the GC box.
Distal regulatory elements can be located thousands of base pairs away from the transcription start site. Enhancers increase transcription in an orientation-independent manner and can act over great distances through DNA looping. Silencers decrease transcription, while insulators block the action of enhancers on unrelated promoters.
Transcription Factor Assembly (Preinitiation Complex for Pol II):
Assembly of the preinitiation complex proceeds in an ordered fashion. First, TFIID (which contains TBP) binds the TATA box. Then TFIIB binds and recruits RNA Pol II along with TFIIF. Next, TFIIE and TFIIH join to complete the preinitiation complex. TFIIH possesses two critical activities: a helicase activity that melts DNA at the start site using ATP, and a kinase activity (CDK7) that phosphorylates the C-terminal domain (CTD) of the largest Pol II subunit. The CTD contains repeats of the heptapeptide YSPTSPS, and phosphorylation of Ser5 promotes promoter clearance and initiation while phosphorylation of Ser2 promotes elongation. Finally, the Mediator complex bridges between gene-specific transcription factors bound at enhancers and the general transcription machinery at the promoter.
<image>A diagram of the eukaryotic transcription initiation complex for RNA Polymerase II. The DNA is shown as a horizontal double helix with labeled regulatory elements: enhancer (far upstream, with an activator protein bound), proximal promoter elements (CAAT box, GC box with Sp1), and the core promoter (TATA box at -25). The assembly of general transcription factors is illustrated step by step: TFIID (with TBP subunit) binds the TATA box, TFIIB bridges to RNA Pol II (recruited with TFIIF), and TFIIE and TFIIH complete the complex. TFIIH is highlighted with its helicase and kinase (CDK7) activities, showing phosphorylation of the CTD tail of Pol II. The Mediator complex is shown as a large multi-subunit bridge connecting an activator at an enhancer (via DNA looping) to the preinitiation complex. An arrow indicates the transition from initiation to elongation upon CTD phosphorylation.</image>
IV. Eukaryotic mRNA Processing
Eukaryotic mRNA undergoes three major processing events that are largely co-transcriptional: 5' capping, 3' polyadenylation, and splicing. These processing steps occur in the nucleus, and the mature mRNA is subsequently exported to the cytoplasm for translation. The CTD of RNA Pol II serves as a platform for recruiting the factors that carry out each of these processing events.
A. 5' Capping
The 5' cap is added co-transcriptionally when the nascent RNA is approximately 20-30 nucleotides long. It consists of a 7-methylguanosine (m7G) linked to the first nucleotide of the RNA via an unusual 5'-5' triphosphate bridge. Three enzymatic steps are required: RNA triphosphatase removes the terminal phosphate from the 5' end, guanylyltransferase adds GMP in a 5'-5' linkage, and methyltransferase adds a methyl group to the N7 of guanine using S-adenosylmethionine (SAM) as the methyl donor.
The 5' cap serves multiple functions. It protects mRNA from 5' exonuclease degradation, thereby increasing mRNA stability. It is required for efficient translation initiation because it is recognized by the initiation factor eIF4E. It also facilitates nuclear export of the mRNA and promotes splicing of the first intron.
B. 3' Polyadenylation
A poly(A) tail consisting of approximately 200 adenine residues is added to the 3' end of the mRNA. The process begins when the polyadenylation signal (AAUAAA) is recognized by CPSF (cleavage and polyadenylation specificity factor). A downstream GU-rich element is recognized by CstF. The pre-mRNA is then cleaved approximately 10-35 nucleotides downstream of AAUAAA, and Poly(A) polymerase (PAP) adds approximately 200 A residues without requiring a template. Poly(A)-binding protein (PABP) subsequently binds the tail.
The poly(A) tail has several important functions. It protects mRNA from 3' exonuclease degradation, increasing stability. It is required for nuclear export and promotes translation initiation through the interaction of PABP with eIF4G, which forms a circular mRNA structure. Importantly, poly(A) tail shortening (deadenylation) is the first step in mRNA degradation.
C. RNA Splicing
Eukaryotic genes contain introns (intervening sequences that are non-coding) and exons (expressed sequences that are coding). Introns must be precisely removed and exons joined in the process of splicing. The average human gene contains approximately 8 introns, and some genes have more than 60. Introns are defined by conserved sequences: the 5' splice site (donor) with a GU dinucleotide at the beginning of the intron, the 3' splice site (acceptor) with an AG dinucleotide at the end, a branch point A residue located approximately 20-50 nucleotides upstream of the 3' splice site, and a polypyrimidine tract between the branch point and the 3' splice site.
The Spliceosome:
The spliceosome is a large ribonucleoprotein complex composed of five snRNPs (small nuclear ribonucleoproteins): U1, U2, U4, U5, and U6. Each snRNP contains a snRNA plus associated proteins, and the snRNAs recognize splice sites by base pairing with the pre-mRNA.
Splicing is a two-step transesterification reaction that requires no net energy input. In Step 1, the 2'-OH of the branch point A attacks the 5' splice site, forming a lariat intermediate in which the branch point A is connected by a 2'-5' phosphodiester bond to the 5' end of the intron, and the 5' exon is released. In Step 2, the free 3'-OH of the 5' exon attacks the 3' splice site, joining the exons together while the lariat intron is released and degraded. The reaction is catalyzed by the RNA components of the spliceosome (the snRNAs themselves), making the spliceosome a ribozyme.
Alternative Splicing:
Different combinations of exons can be included or excluded from the final mRNA, generating multiple mRNA isoforms from a single gene. Approximately 95% of multi-exon human genes undergo alternative splicing, which dramatically increases protein diversity without increasing gene number. For example, the Drosophila DSCAM gene can produce more than 38,000 mRNA variants. Alternative splicing is regulated by splicing regulatory proteins, including SR proteins and hnRNPs, which bind exonic or intronic splicing enhancers or silencers.
<image>A step-by-step diagram of the splicing mechanism. Panel A: A pre-mRNA is shown with two exons (colored boxes) flanking an intron (line). The conserved sequences are labeled: GU at the 5' splice site, the branch point A, the polypyrimidine tract, and AG at the 3' splice site. Panel B: Spliceosome assembly — U1 snRNP binds the 5' splice site, U2 snRNP binds the branch point (forming the A complex), then U4/U5/U6 tri-snRNP joins. After rearrangements, U1 and U4 are released, forming the catalytically active spliceosome. Panel C: The two-step transesterification: Step 1 shows the 2'-OH of the branch point A attacking the 5' splice site, forming the lariat intermediate with the free 5' exon. Step 2 shows the 3'-OH of the 5' exon attacking the 3' splice site, joining the exons and releasing the lariat intron. Panel D: Alternative splicing patterns — exon skipping, alternative 5' or 3' splice sites, intron retention, and mutually exclusive exons are illustrated with different colored exon boxes.</image>
V. Other RNA Processing Events
rRNA Processing:
A large precursor rRNA (45S in humans) is transcribed by RNA Pol I in the nucleolus. This precursor is processed by cleavage and extensive modification, including 2'-O-methylation and pseudouridylation, guided by snoRNAs (small nucleolar RNAs). The result is the mature 28S, 18S, and 5.8S rRNAs. The 5S rRNA is transcribed separately by Pol III.
tRNA Processing:
Precursor tRNAs undergo several processing steps. RNase P cleaves the 5' leader sequence, and this enzyme is notable because it is a ribozyme whose catalytic component is RNA. A 3' endonuclease cleaves the 3' trailer. The CCA-adding enzyme then adds the 3'-CCA sequence (which serves as the amino acid attachment site) without requiring a template. Finally, precursor tRNAs undergo extensive base modifications, including the formation of pseudouridine, dihydrouridine, inosine, and various methylated bases.
VI. Regulation of Gene Expression at the Transcriptional Level
Gene expression is regulated at the transcriptional level through several mechanisms. Transcription factors are proteins that bind DNA regulatory elements such as enhancers, silencers, and promoters, and either activate or repress transcription.
Chromatin remodeling plays a crucial role in controlling access to DNA. Histone acetylation, catalyzed by HATs (histone acetyltransferases), opens chromatin into a euchromatin state that promotes transcription. Conversely, histone deacetylation, catalyzed by HDACs, condenses chromatin into heterochromatin and represses transcription. Histone methylation can either activate or repress transcription depending on the specific residue modified; for example, H3K4me3 is an active mark, whereas H3K27me3 is a repressive mark.
DNA methylation involves the addition of methyl groups to CpG dinucleotides by DNA methyltransferases (DNMTs) and is generally associated with gene silencing, especially when it occurs in promoter CpG islands. DNA methylation is important for X-inactivation, genomic imprinting, and the silencing of transposons. Together, DNA methylation and histone modifications constitute the basis of epigenetics — heritable changes in gene expression that occur without alterations to the DNA sequence itself.
VII. Clinical Correlations
A. Thalassemias and Splicing Mutations
Beta-thalassemia encompasses mutations in the beta-globin gene that reduce or abolish beta-globin synthesis. Many of these mutations affect splice sites or create cryptic splice sites, leading to aberrant splicing that produces unstable or nonfunctional mRNA. Point mutations at the conserved GT (donor) or AG (acceptor) splice site sequences abolish normal splicing entirely.
B. Spinal Muscular Atrophy (SMA)
Spinal muscular atrophy results from loss of the SMN1 gene (survival motor neuron). A groundbreaking treatment, nusinersen (Spinraza), is an antisense oligonucleotide that modifies splicing of the paralog SMN2 to include exon 7, thereby producing functional SMN protein and ameliorating the disease.
C. Systemic Lupus Erythematosus (SLE)
In systemic lupus erythematosus, patients develop autoantibodies against snRNPs (known as anti-Smith antibodies) and other nuclear antigens. Anti-Smith antibodies are highly specific for SLE and serve as an important diagnostic marker.
D. Alpha-Amanitin Poisoning
Ingestion of the Amanita phalloides mushroom introduces alpha-amanitin, which inhibits RNA Pol II and halts mRNA synthesis. This leads to hepatocellular necrosis and renal failure. Symptoms are characteristically delayed 6-12 hours after ingestion and the poisoning can be fatal.
