RNA Synthesis and Processing Study Guide

Functional Roles of RNA and mRNA Vaccines

Ribosomal RNAs (rRNA): These are critical structural and functional components of ribosomes.
Transfer RNAs (tRNA): These molecules play a fundamental role in delivering specific amino acids to the ribosome during protein synthesis.
Messenger RNAs (mRNA): These carry the genetic information that ribosomes utilize for the production of specific protein sequences.
Small Nuclear RNAs (snRNA): These play a specialized role in guiding the splicing of messenger RNAs.
Other RNA Types: * Small regulatory RNAs. * Long noncoding RNAs.
Messenger RNA (mRNA) Vaccines: * These vaccines utilize portions of pathogenic (usually viral) mRNA generated in a laboratory setting. * Mechanism: Human cells injected with these vaccines produce the corresponding viral protein and present it on the cell surface. This process triggers a targeted immune response. * Development: The COVID-19 pandemic significantly accelerated the development, regulatory approval, and public distribution of mRNA vaccines.

Structure and Enzymatic Function of RNA Polymerases

Transcription Functions: During transcription, RNA polymerases (RNAP) perform the following tasks: * Search for initiation sites known as promoter sequences (promoters). * Unwind a short stretch of the double-helical DNA to generate single-stranded DNA templates. * Select the appropriate ribonucleoside triphosphate (NTP) and catalyze the formation of a phosphodiester bond. * Detect specific termination signals that indicate where a transcript must end. * Respond to regulation by activator and repressor proteins that interact with the promoter.
Bacterial RNA Polymerase Subunits (E. coli): * $\alpha$ Subunit: Encoded by the gene rpoA. There are $2$ instances of this subunit, each with a mass of $37\,\text{kDa}$ . * $\beta$ Subunit: Encoded by the gene rpoB. There is $1$ instance with a mass of $151\,\text{kDa}$ . (Color-coded Red in diagrams). * $\beta'$ Subunit: Encoded by the gene rpoC. There is $1$ instance with a mass of $155\,\text{kDa}$ . (Color-coded Blue in diagrams). * $\omega$ Subunit: Encoded by the gene rpoZ. There is $1$ instance with a mass of $10\,\text{kDa}$ . * $\sigma^{70}$ Subunit: Encoded by the gene rpoD. There is $1$ instance with a mass of $70\,\text{kDa}$ .
Catalytic Reaction: * The reaction is identical to that of DNA Polymerase. * RNAPs catalyze the nucleophilic attack of the $3'-OH$ group belonging to the last nucleotide in the chain upon the $\alpha$ -phosphoryl group of the incoming NTP. * This reaction results in the release of a pyrophosphate. * Metal Ion Requirement: RNAPs require two metal ions, typically $Mg^{2+}$ , in the catalytic site. * One $Mg^{2+}$ ion remains tightly bound to the enzyme. * The second $Mg^{2+}$ ion enters with the incoming NTP and departs with the released pyrophosphate.

The Mechanics of Transcription: Initiation, Elongation, and Backtracking

Transcription Bubble: * RNA Polymerase separates a region of the double helix to form the transcription bubble. * This is a complex of double-stranded DNA that has been locally unwound in a region of approximately $17\,\text{base pairs}$ , where the polymerization reactions occur. * Visualization Components: DNA (Blue/Red), RNA (Green), $Mg^{2+}$ (Yellow).
De Novo Chain Formation: * The start site (the first nucleotide) of a coding DNA sequence is denoted as $+1$ . The second is $+2$ , etc. * The nucleotide immediately preceding the start site is denoted as $-1$ . * The very first RNA nucleotide added will be a triphosphate.
Strand Nomenclature: * Template Strand: Has a sequence complementary to the RNA transcript. It is also known as the antisense ( $-$ ) strand. * Coding Strand: Has the same sequence as the RNA transcript, with the exception that Thymine (T) appears in place of Uracil (U). It is also known as the sense ( $+$ ) strand.
Elongation Process: * Step 1: A ribonucleoside triphosphate binds to the active site and forms a Watson–Crick base pair with the DNA template strand. * Step 2: The $3'-OH$ group of the growing RNA chain attacks the $\alpha$ -phosphoryl group of the NTP, forming a phosphodiester bond. * Step 3: The RNA–DNA hybrid translocates, bringing the $3'$ end of the newly added nucleotide into the proper position for the subsequent addition. A part of the RNAP acts as a "wedge" to separate the RNA/DNA hybrid during this movement.
Proofreading and Backtracking: * RNAP can backtrack; however, this is energetically unfavorable because it requires breaking an RNA/DNA base pair. * If an incorrect base is added, hydrogen bonding is incorrect, making backtracking easier. * In the backtracked position, hydrolysis can occur to remove the incorrect base, providing a "good enough" proofreading activity.

Bacterial Promoter Structures and Sigma Factor Diversity

Enzyme Components: * Core Enzyme: The bacterial core RNA polymerase is composed of $\alpha_2\beta\beta'\omega$ . * Holoenzyme: Formed when the $\sigma$ (sigma) subunit joins the core enzyme. The $\sigma$ subunit is essential for finding promoters and participating in initiation.
Promoter Elements in E. coli: * $-10$ element and $-35$ element: Together, these constitute the "core promoter." * Consensus sequences for these elements have been deduced by analyzing many promoters. * Promoters are classified as either STRONG or WEAK. * UP elements: Located $40$ to $60\,\text{bp}$ upstream of the core promoter; these elements bind to the $\alpha$ subunit of RNAP.
Sigma ( $\sigma$ ) Factor Diversity: * Different $\sigma$ factors recognize different promoters, which are associated with genes responding to specific environmental conditions. * Examples: * $\sigma^{70}$ : The default sigma factor for standard growth. * $\sigma^{32}$ : Specifically for the heat shock response. * $\sigma^{54}$ : Specifically for nitrogen starvation conditions.
Unwinding the Helix: * $\sigma$ factor binds to dsDNA, leading to the unwinding of approximately $17\,\text{bp}$ . * Only the $-10$ element is unwound; the $-35$ element remains double-stranded. * The free energy required to break these bonds comes from additional interactions between the template and the bacterial RNAP, which stabilize the "open promoter complex" and help pull the template into the active site.

Transcription Termination Mechanisms in Prokaryotes

Intrinsic Termination: Termination is signaled by the sequence of the RNA produced from the DNA templates.
Types of Signals: * Hairpin-Poly(U): A structural motif in the RNA followed by a string of Uracils. * $\rho$ -dependent (rho-dependent): Requires the rho protein for termination.
In both instances, it is the sequence of the RNA itself that specifies the end of transcription.

Eukaryotic Transcription: Polymerase Types and Compartmentalization

Increased Complexity: Transcription is more highly controlled in Eukaryotes than in Prokaryotes.
Nuclear Membrane: Allows transcription and translation to occur in different cellular compartments (nucleus and cytoplasm, respectively).
Promoter Elements: A wide variety of elements allow for complex transcriptional regulation.
Processing: The degree of post-transcriptional RNA processing is much greater in eukaryotes.
Eukaryotic RNA Polymerase Types: * Type I: Located in the Nucleolus. Transcribes $18S$ , $5.8S$ , and $28S\,\text{rRNA}$ . It is insensitive to $\alpha$ -amanitin. * Type II: Located in the Nucleoplasm. Transcribes mRNA precursors and snRNA. It is strongly inhibited by $\alpha$ -amanitin. * Type III: Located in the Nucleoplasm. Transcribes tRNA and $5S\,\text{rRNA}$ . It is inhibited by high concentrations of $\alpha$ -amanitin.

Post-Transcriptional Processing of Non-Coding RNAs

General Processing: Virtually all eukaryotic transcription products undergo further processing, including splicing, cleavage, base modification, and extension.
Bacterial Modifications: Some prokaryotic transcripts are modified. For example, all bacterial rRNAs are synthesized on the same transcript and then cleaved by RNase III.
Eukaryotic rRNA Processing: * rRNAs are produced on a single transcript. * Ribose and base components undergo extensive modification directed by snoRNPs (small nucleolar ribonucleoproteins). * The pre-rRNA is assembled with ribosomal proteins into a large ribonucleoprotein; subsequent cleavage releases mature rRNAs.
tRNA Processing (RNAP III): * Common to both prokaryotes and eukaryotes: The $5'$ leader is cleaved by RNase P, the $3'$ trailer is removed, and a CCA sequence is added by the CCA-adding enzyme. * Eukaryotic specificities: Moieties on bases and ribose are heavily modified (e.g., methylated bases $X_m$ ; Pseudouridylate $\Psi$ ). Many pre-tRNAs are spliced by an endonuclease and a ligase to remove an intron.

Messenger RNA Processing in Eukaryotes: Capping, Tailing, and Splicing

Key Modifications: Every eukaryotic mRNA receives at least two modifications: * $5'$ Cap: Protects the $5'$ end from phosphatases and nucleases, contributing to stability and enhancing translation by ribosomes. * Poly(A) Tail: A string of approximately $250$ adenine nucleotides added to the $3'$ end by poly(A) polymerase after cleavage by an endonuclease. Enhances stability and translation efficiency.
Exons and Introns: * Exons: Sequences that contain protein-coding information. * Introns: Intervening sequences (ranging from $50$ to $10,000\,\text{nt}$ ) that must be removed. * RNA Splicing: The process of removing introns and linking exons, catalyzed by the spliceosome.
Intron Motifs and Consensus Sequences: * Introns typically begin with $GU$ and end with $AG$ . * $5'$ Splice Site (Vertebrates): Consensus sequence is AGGUAAGU, where the first GU marks the intron start. * $3'$ Splice Site: Consists of a polypyrimidine tract ( $10$ pyrimidines, $C/T$ ), followed by the sequence NCAG, where AG marks the intron end. * Branch Site: Located $20$ to $50$ nucleotides upstream of the $3'$ splice site.
Splicing Mechanism: * Consists of two transesterification reactions. * The first reaction involves the $2'-OH$ of an adenylate at the branch site attacking the phosphodiester linkage between exon 1 and the $5'$ end of the intron. * The second reaction involves the $3'-OH$ of exon 1 attacking the linkage between the intron and exon 2, joining the exons. * The released lariat structure is recognized as waste and degraded in the nucleus.

Clinical Pathology and Diseases of RNA Splicing and Editing

Splicing Mutations: Estimates suggest splicing defects cause at least $15\%$ of all genetic diseases. Mutations can be cis-acting (in the pre-mRNA) or trans-acting (in splicing factors).
Thalassemias: Conditions of defective hemoglobin synthesis caused by mutations at splice sites in the introns or exons of the hemoglobin $\beta$ chain.
Specific Genetic Disorders and Linked Genes: * Acute intermittent porphyria: Porphobilinogen deaminase. * Breast and ovarian cancer: BRCA1. * Cystic fibrosis: CFTR. * Frontotemporal dementia: $\tau$ (tau) protein. * Hemophilia A: Factor VIII. * HGPRT deficiency (Lesch-Nyhan syndrome): Hypoxanthine-guanine phosphoribosyltransferase. * Leigh encephalomyelopathy: Pyruvate dehydrogenase E1 $\alpha$ . * Severe combined immunodeficiency (SCID): Adenosine deaminase. * Spinal muscle atrophy: SMN1 or SMN2.
RNA Editing: * Post-transcriptional changes in nucleotide sequence. A single nucleotide change can create a new codon or a stop codon. * Example: Enzyme-catalyzed deamination of a cytidine residue in the mRNA for apolipoprotein B-100 changes a glutamine codon (CAA) to a stop codon (UAA).

Ribozymes: The Catalytic Capacity of RNA

Nature of Ribozymes: Specific classes of RNA molecules that adopt complex, specific shapes to act as enzymes.
Example: The $23S\,\text{rRNA}$ forms intricate shapes to perform catalytic functions.
Self-Splicing Introns: * These introns can excise themselves without external help. * Initially identified in rRNA from Tetrahymena (a unicellular eukaryote). * Requires guanosine as a cofactor. * Catalysis is possible purely through the structure adopted by the RNA molecule itself.