Chapter 6 RNA Synthesis and Processing

6.1 Transcription in Prokaryotes

• Transcription is the first step in gene expression and a key point of regulation.

• In eukaryotes, RNAs are modified, including intron removal via splicing.

• Transcription is the synthesis of RNA from DNA, catalyzed by RNA polymerases.

Types of RNA Molecules

• mRNA: Carries genetic information from DNA to ribosomes.

• tRNA: Adapters between amino acids and mRNA codons.

• rRNA: Structural and catalytic components of ribosomes.

• snRNA: Structural components of spliceosomes.

• miRNA: Short RNAs that block expression of complementary mRNAs.

Transcription in Prokaryotes

• E. coli was instrumental in mRNA discovery and RNA polymerase study.

• Experiments in E. coli revealed transcription regulation mechanisms.

• Regulated gene expression enables responses to environmental changes like nutrient availability.

RNA Polymerase and RNA Synthesis

• RNA polymerase is the primary enzyme for RNA synthesis.

• It catalyzes the polymerization of ribonucleotide triphosphates (NTPs) using a DNA template.

• RNA synthesis is similar to DNA synthesis, proceeding in the 5' to 3' direction.

• RNA polymerase doesn't require a primer to initiate RNA synthesis; it starts de novo at specific gene start sites.

Structure of E. coli RNA Polymerase

• RNA polymerase is a complex enzyme made of multiple polypeptide chains.

• Subunit types include α, β, β′, ω, and σ.

• The σ subunit is loosely bound and can be separated; the core polymerase (2α, β, β′, ω) can catalyze RNA synthesis without it.

• The σ subunit is crucial for recognizing specific DNA sequences that initiate transcription.

• The holoenzyme consists of all subunits bound together (α2ββ'ωσ).

Transcription Cycle

• Initiation: RNA polymerase holoenzyme binds to the promoter, unwinds DNA, and forms phosphodiester bonds between 7 to 12 nucleotides; requires sigma factor (σ).

• Elongation: (σ) dissociates, and the core elongates RNA with high processivity.

• Termination: Polymerase dissociates from template DNA and releases new RNA; often uses rho protein (⍴).

• RNA synthesis involves three steps: initiation, elongation, and termination.

E. coli Promoters

• Promoters are DNA sequences where RNA polymerase binds to initiate transcription.

• Identified through nucleotide sequence comparisons of genes from E. coli.

• Common elements: -10 and -35 regions, located approximately 10 and 35 base pairs upstream of the transcription start site.

• Genes with consensus promoters are transcribed more efficiently.

• RNA polymerase binds to a 60-base-pair region, from -40 to +20.

• The σ subunit specifically binds to -35 and -10 regions.

Promoter Function

• σ directs polymerase to promoters by specifically binding to -35 and -10 sequences.

• Initiates transcription at the gene start.

Promoter Complexes

• Closed-promoter complex: Initial binding, DNA remains unwound.

• Open-promoter complex: DNA unwound, single-stranded DNA template is available.

• The polymerase unwinds 12-14 bases of DNA (-12 to +2), making single-stranded DNA available for transcription.

• Transcription initiation involves the joining of two free NTPs.

Transcription by E. coli RNA Polymerase Steps

1. Polymerase binds nonspecifically to DNA.

2. Specific binding of σ to -35 and -10 promoter sequences.

3. Formation of closed-promoter complex.

4. Formation of open-promoter complex with DNA unwinding.

5. Initiation of transcription.

6. Release of σ.

7. Elongation of RNA.

Transcription Termination

• Transcription termination occurs in two ways:

  • Rho-independent termination (intrinsic): The DNA of a terminator sequence encodes an inverted repeat and an adjacent stretch of uracils.

  • Rho-dependent termination: Involves a terminator sequence and a rho factor or protein.

Rho-Dependent Termination

• Rho protein (ρ) is required for termination and occurs in prokaryotes.

• Rho binds to a specific site on the mRNA called the Rho Utilization (rut) site.

• Rho travels along the mRNA in the 5' to 3' direction using ATP hydrolysis.

• When RNA polymerase stalls at a terminator sequence, Rho catches up and causes RNA polymerase to release the mRNA.

• Termination depends on both the terminator sequence and Rho protein activity.

Transcription Termination Signals

• Termination is signaled by a GC-rich inverted repeat followed by seven A residues.

• The inverted repeat forms a stable stem-loop structure in the RNA, terminating transcription and leading to dissociation of the RNA from the DNA template.

6.2 Transcription in Eukaryotes

Eukaryotic vs. Bacterial Transcription

• Bacterial transcription: Single core RNA polymerase, relatively simpler process.

• Eukaryotic transcription: Three nuclear RNA polymerases, greater complexity.

Polymerases of Eukaryotes

Eukaryotic RNA Polymerase Complex

• Each nuclear RNA polymerase is a complex enzyme with 12–17 different subunits.

• Shares common features with bacterial RNA polymerase.

• All three eukaryotic RNA polymerases have nine conserved subunits.

• Five subunits are related to α, β, β′, and ω subunits of bacterial RNA polymerase.

• Yeast RNA Polymerase II structure resembles the bacterial enzyme, suggesting conserved transcription mechanisms across domains.

RNA Polymerase II & Transcription in Eukaryotes

• Involved in transcription from most Pol II promoters.

• Forms part of the basic transcription machinery.

• Regulates gene expression by binding to specific DNA sequences and controlling individual gene expression (Gene-Specific Transcription Factors).

• Pol II activity differs from bacterial RNA polymerase; eukaryotic transcription requires additional proteins for initiation (Transcriptional factors).

• Unlike bacterial σ factors, these factors are not inherently associated with the polymerase.

Promoter Elements in RNA Polymerase II Transcription

• Promoters of RNA Polymerase II genes have diverse sequence elements around the initiation site.

• TATA box, resembling the bacterial -10 element, was initially identified.

• Genome-wide studies show TATA boxes in only 10-20% of RNA Pol II promoters.

• Additional elements include Initiator (Inr), TFIIB Recognition Elements (BRE), and elements downstream of the transcription start site (DCE, MTE, and DPE).

General Transcription Factors for RNA Pol II Initiation

• Five general transcription factors are needed for RNA Pol II initiation.

• First step: TFIID binding to the promoter.

• TFIID comprises TATA-binding protein (TBP) and TBP associated factors (TAFs).

• TBP binds the TATA box; TAFs bind Inr, DCE, MTE, and DPE sequences.

Formation of Transcription Complex

• TFIIB is recruited after TFIID binding and binds TBP and BRE sequences.

• TFIIB acts as a bridge to RNA Polymerase II.

• RNA Pol II binds the TBP-TFIIB complex along with TFIIF.

• Additional Factors: TFIIE and TFIIH bind and complete the preinitiation complex.

Roles of TFIIH and C-Terminal Domain (CTD)

1. Roles of TFIIH

   • Helicases: Unwind DNA around the initiation site.

   • Protein Kinase: Phosphorylates the C-terminal domain of RNA Pol II.

2. C-terminal Domain (CTD) of RNA Polymerase II

   • Comprises tandem repeats of seven amino acids.

   • Consensus Sequence: Tyr-Ser-Pro-Thr-Ser-Pro-Ser.

   • Phosphorylation of Serine-5 (fifth amino acid) by TFIIH protein kinase.

Transcription Factors

Mediator Complex in Transcription

• Comprises over 20 distinct subunits.

• Interacts with both general transcription factors and RNA polymerase.

• Stimulates basal transcription and connects general and gene-specific factors.

RNA Polymerase II/Mediator Complexes and Transcription Initiation

• The Mediator complex binds to the non-phosphorylated C-terminal domain (CTD) of polymerase II and is released following phosphorylation of the CTD when transcription initiates.

• The phosphorylated CTD then binds elongation and processing factors that facilitate mRNA synthesis and processing.

Regulation of Transcription in Eukaryotes

• More complicated than in prokaryotes.

• Chromatin limits access of regulatory proteins to promoters; factors must reorganize the chromatin.

• In addition to promoters, eukaryotic genes have ‘enhancers,’ also known as upstream activation sequences.

• DNA looping permits multiple proteins to bind to multiple DNA sequences.

Transcription Elongation

• Transcription can also be regulated at elongation.

• Many genes have molecules of RNA polymerase II that have started transcription but are stalled immediately downstream of promoters.

• Following initiation, the polymerase pauses within about 50 nucleotides due to negative regulatory factors, including NELF (negative elongation factor) and DSIF.

• Continuation depends on another factor: P-TEFb (positive transcription-elongation factor-b).

Transcription Termination

• Protein-coding genes have a polyadenylation signal at their 3′ end.

• Thr-4 of RNA polymerase II C-terminal domain (CTD) is phosphorylated during transcription of the 3′ end.

• Phosphorylation recruits an RNA endonuclease that cleaves the polyadenylation signal in the nascent transcript.

• This releases the mature mRNA.

• The remaining nascent transcript's new 5′ end is degraded by a 5′ to 3′ exonuclease.

• This action dislodges the polymerase from the DNA template.

Transcription by RNA Polymerases I

• RNA Polymerase I:

  • Responsible for transcribing ribosomal RNA genes.

  • Produces high levels of rRNAs for protein synthesis.

  • Active in the nucleolus.

  • Promoters (elements CPE and UCE) are recognized by UBF and SL1 transcription factors.

Transcription by RNA Polymerases III

• RNA Polymerase III:

  • Transcribes 5S rRNA, tRNAs, and some small nuclear RNAs.

  • Uses distinct promoters, some within the transcribed sequence.

  • TFIIIA initiates assembly, followed by TFIIIC, TFIIIB, and the polymerase.

6.3 RNA Processing and Turnover

• rRNAs and tRNAs undergo processing in both bacteria and eukaryotic cells.

• Eukaryotic mRNA primary transcripts undergo extensive modifications.

Processing of Ribosomal RNA (rRNA)

• Prokaryotes:

  • Two subunits: 50S and 30S.

  • Three ribosomal RNAs (23S, 16S, 5S) equivalent to eukaryotic 28S, 18S, and 5S rRNAs, processed from a single pre-rRNA.

• Eukaryotes:

  • Two subunits: 60S and 40S.

  • Four species of ribosomal RNAs (5.8S, 18S, 28S) are derived from a single pre-rRNA transcript.

  • 5S rRNA in eukaryotes is transcribed separately and requires less processing.

Processing of rRNA involves

• Cleavage of precursor transcript.

• Addition of methyl groups to specific ribose residues.

• Conversion of uridine to pseudouridine.

• Processing Machinery:

  • Involves proteins and RNAs localized in the nucleolus.

  • Nucleoli contain >300 proteins and ~200 small nucleolar RNAs (snoRNAs).

  • snoRNAs complex with proteins, forming snoRNPs.

  • Two families of snoRNAs associated with proteins catalyzing ribose methylation or pseudouridine formation: Methyltransferase snoRNPs and Uridine isomerase snoRNPs.

Processing of Transfer RNA (tRNA)

1. Cleavage at the 5' end of the tRNA is catalyzed by the RNase P ribozyme; cleavage at the 3' end is catalyzed by a conventional protein RNase.

2. CCA terminus is then added to the 3' end of many tRNAs in a post-transcriptional processing step.

3. Finally, some bases are modified at characteristic positions in the tRNA molecule.

Structure of tRNA Modified Bases

• Dihydrouridine and ribothymidine are formed by modification of uridines in tRNA.

• Inosine and methylguanosine are formed by the modification of guanosines.

mRNA Processing: Bacteria vs. Eukaryotes

mRNA Processing in Eukaryotes

• mRNA molecules are associated with proteins as messenger ribonucleoprotein particles (mRNPs).

• RNA Polymerase II's C-terminal domain (CTD) is pivotal:

  • Serves as a binding site for processing enzyme complexes.

  • Ensures specificity in mRNA processing.

  • Coupled to transcription for coordination.

• mRNA processing includes:

  • Modification of both ends of the initial transcript.

  • Removal of introns from the middle.

5' End Modification - 7-methylguanosine Cap

• Enzymes are recruited to the phosphorylated CTD post-transcription initiation.

• The cap is added after transcribing the first 20-30 nucleotides.

• Process:

  • Addition of guanosine triphosphate (GTP) in reverse orientation.

  • Methylation of G residue and ribose moieties of 5' nucleotides.

• Functions of the 5' cap:

  • Stabilizes the RNA.

  • Aligns eukaryotic mRNAs on the ribosome during translation.

3’ End Modification - Polyadenylation

• 3' end processing in eukaryotic mRNAs involves polyadenylation.

• Signals for polyadenylation:

  • Conserved hexanucleotide (AAUAAA in mammals), 10–30 nucleotides upstream of the polyadenylation site.

  • U- or GU-rich downstream sequence element.

• Polyadenylation process:

  • Recognition of polyadenylation signals.

  • Endonuclease cleaves the RNA chain.

  • Poly-A polymerase adds a poly-A tail (about 200 nucleotides).

• Recognition of polyadenylation signal leads to:

  • Termination of transcription.

  • Cleavage and polyadenylation of the mRNA.

  • Degradation of RNA synthesized downstream of the poly-A addition site.

Splicing

• In Vitro Systems Enable Insight:

  • Pre-mRNA splicing understanding advanced with in vitro systems.

  • Cloning structural genes with introns adjacent to viral RNA polymerase promoters facilitated pre-mRNA synthesis.

Splicing Mechanism

• Two-Step Splicing Process

  • Step 1: Cleavage at 5' Splice Site:

    • 5' end of intron joined to an adenine nucleotide within the intron.

    • Forms a unique bond between the intron and adenine.

    • Produces a lariat-like structure.

  • Step 2: Cleavage at 3' Splice Site and Exon Ligation:

    • Simultaneous cleavage at 3' splice site.

    • Ligation of two exons.

    • Intronic lariat structure is excised, linearized, and degraded within the nucleus.

Spliceosomes

• Splicing is accomplished with the help of spliceosomes, which remove introns from the genes in RNA.

• A spliceosome is a large and complex molecular machine found primarily within the nucleus of eukaryotic cells.

• The spliceosome is assembled from small nuclear RNAs (snRNA) and approximately 80 proteins.

• The RNA components of the spliceosome are 5 types - U1, U2, U4, U5, and U6.

• These snRNAs form small nuclear ribonucleoprotein particles (snRNPs).

Assembly of the Spliceosome

• The first step in spliceosome assembly is the binding of U1 snRNP to the 5' splice site (SS), followed by the binding of U2 snRNP to the branch point.

• A preformed complex consisting of U4/U6 and U5 snRNPs then enters the spliceosome.

• U4 and U1 dissociate from the spliceosome.

• U2, U5, and U6 catalyze both formation of the lariat-like intermediate and ligation of the exons.

Self Splicing RNAs

Self-Splicing Introns

• Found in mitochondria, chloroplasts, and bacteria.

• Two groups of self-splicing introns:

  • Group I—cleavage at 5′ SS is mediated by a guanosine cofactor.

  • Group II—cleavage of 5′ SS results from attack by an adenosine nucleotide in the intron.

Alternative Splicing

• Splicing is crucial in mRNA processing and offers a means of gene expression regulation.

• Alternative splicing generates multiple mRNA variants and, consequently, various proteins from a single pre-mRNA.

• Nearly all human genes undergo alternative splicing, vastly increasing protein diversity.

• This process is a key mechanism for tissue-specific and developmental gene expression regulation.

Example: Sex Determination in Drosophila

• The transformer (tra) gene's pre-mRNA undergoes alternative splicing to determine sex.

• Mechanism:

  • Three exons in the tra pre-mRNA.

  • Different 3′ splice sites are used in males and females.

  • Male: U2AF splicing factor binds, selecting the upstream site.

  • Female: SXL protein blocks U2AF, favoring the downstream site.

• Consequences:

  • Male mRNA: Contains a termination codon (UAG), so no protein is produced.

  • Female mRNA: Produces functional transformer protein, pivotal in sex determination.

Alternative Splicing: Dscam Gene

• Dscam gene in Drosophila can generate over 38,000 distinct mRNAs, impacting neuron connections in brain development.

Muscular Dystrophy

• DMD Gene and Dystrophin:

  • The DMD gene is extensive (2.4 Mb) with 79 exons.

  • Encodes dystrophin, a critical protein for muscle stability and contraction.

• DMD Mutations:

  • Mutations cause premature termination of dystrophin translation.

  • Deletions lead to frameshifts, resulting in a non-functional protein (DMD).

  • In-frame deletions lead to a partially functional protein (Becker MD).

• Exon Skipping Therapy:

  • Targets specific exon(s) to restore the reading frame.

  • Prevents inclusion of problematic exons in mRNA.

  • Enables the production of a partially functional dystrophin.

• Exondys 51:

  • First FDA-approved drug for DMD treatment in 2016.

  • Targets exon 51 skipping, developed by Sarepta Therapeutics.

RNA Editing

• RNA editing modifies the nucleotide sequence of RNA after transcription.

• It's vital for cellular function and occurs in various organisms.

• This process creates diverse protein versions with unique functions.

• Unlike splicing, which removes RNA sections, editing alters individual nucleotides.

• There are two generic classes of RNA editing in nuclei:

  • Substitution editing: enzymatic deamination of either Cytosine-to-Uridine or Adenosine-to-Inosine nucleotides.

  • Addition/deletion editing: Multiple U residues are inserted.

Apolipoprotein B mRNA Editing

• The liver produces unedited Apo-B100 (4536 amino acids), while the intestine generates edited Apo-B48 (2152 amino acids).

• Editing converts a C to U, changing the glutamine codon to a stop codon, resulting in a shorter protein.

• Tissue-specific editing yields structurally and functionally distinct proteins.

A-to-I Editing in the Nervous System

• Crucial in the nervous system, leading to single amino acid changes in neuronal ion channels and receptors.

• Example: Serotonin receptor mRNAs are edited at multiple sites, resulting in various receptor versions with distinct signaling functions.

• Significance in Neurology:

  • Mutants lacking the editing enzyme in model organisms exhibit neurological defects (e.g., C. elegans, Drosophila, and mice).

mRNA Degradation

• RNA Stability:

  • Ribosomal and transfer RNAs are highly stable (>90% of cellular RNA).

  • Bacterial mRNAs have short half-lives (2-3 minutes) for rapid response to environmental changes.

  • Eukaryotic mRNAs exhibit varying degradation rates, contributing to gene expression regulation.

  • Mammalian mRNA half-lives range from < 30 mins to ~20 hours.

• Degradation Initiation:

  • mRNA ends are protected by capping (5') and polyadenylation (3').

  • Cytoplasmic degradation starts with poly-A tail shortening.

Degradation Pathways

• Poly-A Tail Shortening / Deadenylation-Dependent Pathway:

  • Deadenylated mRNAs are degraded by nucleases from the 3' end or via 5' cap removal followed by 5' end degradation.

• Regulatory Elements:

  • AU-rich sequences near 3' ends can stabilize or target mRNAs for degradation.

  • RNA-binding proteins, influenced by extracellular signals, modulate degradation rates.

• miRNA Regulation:

  • miRNAs participate in mRNA degradation and translation inhibition.

• Endonucleolytic Cleavage:

  • Endonucleases can cleave mRNA within the coding region, leading to its degradation.