RNA Synthesis (Chapter 29)

Types and Functional Roles of RNA Molecules

• RNA molecules serve a wide variety of essential biological functions, ranging from structural components to information carriers and regulatory elements:     - Ribosomal RNAs (rRNAs): These are critical structural and functional components of ribosomes, the cellular machinery responsible for protein synthesis.     - Transfer RNAs (tRNAs): These molecules play a vital role in protein synthesis by delivering specific amino acids to the ribosome.     - Messenger RNAs (mRNAs): These carry the genetic information required for ribosomes to produce specific protein sequences.     - Small nuclear RNAs (snRNAs): These are involved in the guidance and execution of the splicing process for messenger RNAs.     - Other RNAs: This category includes small regulatory RNAs and long noncoding RNAs (lncRNAs), which manage various cellular processes.

• Medical Application: Messenger RNA (mRNA) Vaccines:     - These vaccines utilize lab-generated portions of pathogenic (typically viral) mRNA.     - Mechanism: When human cells are injected with these vaccines, they produce the corresponding viral protein and present it on the cell surface. This process triggers a robust immune response.     - The global COVID-$19$ pandemic significantly accelerated the development, regulatory approval, and public distribution of mRNA vaccine technology.

Characteristics and Catalytic Mechanisms of RNA Polymerase (RNAP)

• RNA polymerases are the primary enzymes responsible for catalyzing the synthesis of RNA during the process of transcription.

• Core Functions of RNA Polymerases during Transcription:     - Searching for initiation sites: They identify specific DNA segments called promoter sequences (or promoters).     - DNA Unwinding: They unwind a short stretch of the double-helical DNA to create single-stranded DNA templates.     - Nucleotide Selection: They identify and select the correct ribonucleoside triphosphate (NTP) to incorporate into the growing chain.     - Catalysis: They catalyze the formation of phosphodiester bonds.     - Termination Detection: They recognize specific termination signals that indicate where a transcript should end.     - Regulation: Their activity is governed by interaction with activator and repressor proteins that bind at or near the promoter.

• Bacterial RNA Polymerase Subunit Composition:     - $\alpha$ (rpoA): $2$ subunits, mass of $37\,kDa$ each.     - $\beta$ (rpoB): $1$ subunit, mass of $151\,kDa$. (Represented in red in structural models).     - $\beta'$ (rpoC): $1$ subunit, mass of $155\,kDa$. (Represented in blue in structural models).     - $\omega$ (rpoZ): $1$ subunit, mass of $10\,kDa$.     - $\sigma^{70}$ (rpoD): $1$ subunit, mass of $70\,kDa$.

• The Chemical Reaction of RNA Synthesis:     - This reaction is chemically identical to the reaction catalyzed by DNA polymerase (DNAP).     - RNA polymerases catalyze a nucleophilic attack handled by the $3'-OH$ group of the last nucleotide in the growing chain upon the $\alpha\text{-phosphoryl}$ group of the incoming NTP.     - This reaction results in the release of a pyrophosphate ($PP_i$).     - Requirement for Metal Ions: RNAP requires two metal ions, typically $Mg^{2+}$ (Magnesium), in the catalytic site:         - One $Mg^{2+}$ ion remains tightly bound to the enzyme.         - The second $Mg^{2+}$ ion enters the complex with the incoming NTP and departs along with the pyrophosphate.

The Transcription Bubble and Chain Elongation

• The Transcription Bubble:     - RNA polymerase separates a region of the DNA double helix to form what is known as the "transcription bubble."     - Definition: A complex of double-stranded DNA that has been locally unwound in a region spanning approximately $17$ base pairs where actual polymerization occurs.     - Structural components involve DNA, nascent RNA, and $Mg^{2+}$ ions.

• RNA Chain Formation and Conventions:     - RNA chains are formed de novo, meaning they do not require a primer.     - The start site (the first nucleotide) of a coding DNA sequence is denoted as $+1$, the second as $+2$, and so on.     - The first RNA nucleotide synthesized will retain a triphosphate group at its $5'$ end.     - The nucleotide immediately preceding the start site in the DNA is denoted as $-1$.     - Template vs. Coding Strands:         - Template Strand: Has a sequence complementary to the RNA transcript. Also referred to as the antisense ($-$) strand.         - Coding Strand: Has the same sequence as the RNA transcript (with the exception of T being replaced by U in RNA). Also referred to as the sense ($+$) strand.

• Steps of RNA Elongation:     - Step 1: A ribonucleoside triphosphate (NTP) binds in the RNA polymerase active site, forming a Watson–Crick base pair with the template DNA strand.     - Step 2: The $3'-OH$ group of the growing RNA chain performs a nucleophilic attack on the $\alpha\text{-phosphoryl}$ group of the NTP, forming a phosphodiester bond.     - Step 3: The RNA–DNA hybrid translocates. This movement brings the $3'$ end of the newly added nucleotide into the proper position for the next addition. A specific part of RNAP acts as a "wedge" to separate the RNA/DNA hybrid during this movement.

• Translocation Logistics:     - Translocation requires no external energy.     - Visualizing the bubble: As the polymerase moves, it performs unwinding at the front (downstream) and rewinding at the back (upstream).

• Backtracking and Proofreading:     - RNAP can backtrack. Normally, this is thermodynamically unfavorable because it requires breaking RNA/DNA base pairs.     - If an incorrect base is added, backtracking becomes easier because the hydrogen bonding is inherently weak or incorrect.     - Once in the backtracked position, hydrolysis of the RNA can occur, allowing the incorrect segment to be removed. This serves as a "good enough" proofreading mechanism for RNAP.

Transcriptional Initiation and Bacterial Promoters

• RNAP Holoenzyme vs. Core Enzyme:     - Core Enzyme: The bacterial structure composed of $\alpha_2\beta\beta'\omega$.     - $\sigma$ Subunit: Responsible for helping the enzyme locate promoter sites and participating in the initiation of synthesis.     - Holoenzyme: Formed when the $\sigma$ subunit joins the core enzyme structure.

• Promoter Elements in E. coli:     - $-10$ Element: A sequence located approximately $10$ bp upstream of the start site.     - $-35$ Element: A sequence located approximately $35$ bp upstream of the start site.     - Core Promoter: Comprised of the $-10$ and $-35$ elements together. Consensus sequences for these elements have been determined by analyzing numerous different promoters.     - UP Elements: Found $40$ to $60$ bp upstream of the core promoter; these bind specifically to the $\alpha$ subunit of RNAP.     - Promoters vary in strength, being classified as either strong or weak.

• Diversity of $\sigma$ Factors:     - Different $\sigma$ factors recognize different promoter sequences, allowing the cell to regulate groups of genes in response to specific environmental conditions:         - $\sigma^{70}$: The default $\sigma$ factor used for most genes.         - $\sigma^{32}$: Used during the heat shock response.         - $\sigma^{54}$: Used during nitrogen starvation.

• The Initiation Process:     - The $\sigma$ factor binds to double-stranded DNA (dsDNA), then approximately $17\,bp$ are unwound.     - The $-10$ element is unwound, while the $-35$ element remains intact.     - The free energy required to break the DNA bonds comes from compensatory interactions between the DNA template and the bacterial RNAP. These interactions stabilize the open promoter complex and pull the template strand into the active site.

Transcriptional Termination

• RNA synthesis is terminated by the properties of the RNA sequence produced, not just DNA sequences.

• Intrinsic Termination Signals:     - Hairpin-Poly(U): The RNA forms a hairpin structure followed by a sequence of Uracil residues. This structure causes the RNA to dissociate from the DNA template.     - $\rho\text{-dependent}$ (rho-dependent): Relies on the $\rho$ protein factor to assist in termination.

• In both mechanisms, the sequence of the RNA itself serves as the signal to stop transcription.

Transcription in Eukaryotes

• Transcription in eukaryotes is significantly more complex and highly controlled than in prokaryotes.

• Key Differences in Eukaryotes:     - Nuclear Membrane: Transcription and translation are spatially separated (transcription in the nucleus, translation in the cytoplasm).     - Regulatory Complexity: A vast array of promoter elements allows for intricate control over gene expression.     - Extensive Processing: Eukaryotic RNA undergoes much more modification than prokaryotic RNA.

• The Three Eukaryotic RNA Polymerases:     - Type I: Located in the nucleolus. Transcribes $18S$, $5.8S$, and $28S\,rRNA$. It is insensitive to $\alpha\text{-amanitin}$.     - Type II: Located in the nucleoplasm. Transcribes mRNA precursors and snRNA. It is strongly inhibited by $\alpha\text{-amanitin}$.     - Type III: Located in the nucleoplasm. Transcribes tRNA and $5S\,rRNA$. It is inhibited by high concentrations of $\alpha\text{-amanitin}$.

RNA Processing and Modification

• General Processing Mechanisms:     - Virtually all eukaryotic transcripts undergo processing: splicing, cleavage, base modification, or extension.     - While more common in eukaryotes, some prokaryotic transcripts are modified. For example, all bacterial rRNAs are synthesized on a single transcript and then cleaved by RNase III.

• Processing of Ribosomal RNA (rRNA):     - Eukaryotes produce rRNAs on a single large transcript.     - Ribose and base components are extensively modified, guided by RNA-protein complexes called small nucleolar ribonucleoproteins (snoRNPs).     - Pre-rRNA is assembled with ribosomal proteins to form a large ribonucleoprotein; mature rRNAs are released following specific cleavage events.

• Processing of Transfer RNA (tRNA):     - RNAP III initiates tRNA production in eukaryotes. Processing steps include:         - Cleavage of the $5'$ leader by RNase P.         - Removal of the $3'$ trailer.         - Addition of the CCA sequence by the CCA-adding enzyme.         - Base and ribose moieties are heavily modified (e.g., methylation represented as $Xm$ or pseudouridylation represented as $\Psi$).         - Many eukaryotic pre-tRNAs contain an intron that is removed by an endonuclease and a ligase.

• Modification of Eukaryotic Messenger RNA (mRNA):     - $5'$ Cap: Protects the $5'$ end from phosphatases and nucleases. It contributes to mRNA stability and enhances translation efficiency.     - Poly(A) Tail: A string of approximately $250$ adenine nucleotides added to the $3'$ end by poly(A) polymerase after cleavage by an endonuclease. It enhances stability and translation efficiency.

Splicing: The "Secret Sauce" of Eukaryotic mRNA

• Eukaryotic genes consist of Exons (protein-coding information) and Introns (intervening non-coding sequences).

• Introns can range from $50$ to $10,000$ nucleotides in length.

• Splicing Definition: The process where introns are removed and exons are joined to form the final mRNA.

• The Spliceosome: A complex of RNA and protein that catalyzes the splicing reaction.

• Common Motifs in Introns:     - Introns typically begin with GU and end with AG.     - $5'$ Splice Site: In vertebrates, the consensus sequence is $AGGUAAGU$ (the first GU is the start of the intron).     - $3'$ End of Intron: Consists of a polypyrimidine tract (approximately $10$ pyrimidines, C or T) followed by the sequence $NCAG$ (where AG indicates the end of the intron).     - Branch Site: Located $20$ to $50$ nucleotides upstream of the $3'$ splice site.

• Two-Step Transesterification Process:     1. Cleavage occurs at the phosphodiester linkage between exon $1$ and the $5'$ end of the intron. The attacking group is the $2'-OH$ of an adenylate residue located at the branch site.     2. The resulting $3'-OH$ of exon $1$ then attacks the phosphodiester linkage between the intron and exon $2$. This joins the two exons and releases the intron as a lariat structure.     - The lariat is recognized as waste and degraded within the nucleus.

• Clinical Significance of Splicing:     - Splicing defects are estimated to cause at least $15\%$ of all genetic diseases. Mutations can be cis-acting (in the pre-mRNA) or trans-acting (in splicing factors).     - Thalassemias: Caused by mutations at splice sites in the hemoglobin $\beta$ chain introns or exons, often causing a stop codon that would normally be removed to be retained.     - Splicing-Related Disorders:         - Acute intermittent porphyria (Porphobilinogen deaminase)         - Breast and ovarian cancer (BRCA1)         - Cystic fibrosis (CFTR)         - Frontotemporal dementia ($\tau$ protein)         - Hemophilia A (Factor VIII)         - HGPRT deficiency / Lesch-Nyhan syndrome (Hypoxanthine-guanine phosphoribosyltransferase)         - Leigh encephalomyelopathy (Pyruvate dehydrogenase E1 $\alpha$)         - Severe combined immunodeficiency (Adenosine deaminase)         - Spinal muscle atrophy (SMN1 or SMN2)

RNA Editing and Ribozymes

• RNA Editing:     - Post-transcriptional changes to the nucleotide sequence of RNA.     - Even a single nucleotide change can create a new codon or a stop codon.     - Example: In apolipoprotein B-$100$ mRNA, enzyme-catalyzed deamination of a cytidine residue changes a glutamine codon (CAA) to a stop codon (UAA).

• Ribozymes (Catalytic RNA):     - Ribozymes are RNA molecules that adopt specific three-dimensional shapes to act as enzymes. Shape determines function.     - Example: The $23S\,rRNA$ forms intricate shapes necessary for catalysis.

• Self-Splicing Introns:     - Some RNA molecules can catalyze their own excision.     - These were first identified in the rRNA of Tetrahymena (a unicellular eukaryote).     - This process requires guanosine as a cofactor and relies on the intron adopting a specific structural configuration.