Transcription and RNA Processing

The Central Dogma and Information Flow

The fundamental pathway of biological information flow is described by the Central Dogma: $DNA \rightarrow RNA \rightarrow Protein$ .
DNA Replication: The process by which $DNA$ makes a copy of itself ( $DNA \rightarrow DNA$ ), catalyzed by $DNA Polymerase$ .
Transcription: The synthesis of $RNA$ from a $DNA$ template ( $DNA \rightarrow RNA$ ), catalyzed by $RNA Polymerase$ .
Translation: The decoding of $RNA$ into a sequence of amino acids to form a protein ( $RNA \rightarrow Protein$ ), occurring at the $Ribosome$ .
Heritability: Watson and Crick noted that the specific pairing of nitrogenous bases suggests a possible copying mechanism for genetic material. DNA is the heritable material of life.
Protein Function: For proteins, structure equals function. The $3D$ shape of a protein dictates its activity. This shape is determined by the specific sequence of amino acids, which is ultimately dictated by the $DNA$ sequence.

Gene Expression and Amplification

Definition of Expression: A gene is considered "expressed" when the information within the $DNA$ affects the cell's properties and activities (e.g., when a protein-coding gene results in a functional protein).
Gene to Protein Relationship: In eukaryotic cells, one gene typically contains instructions for building one protein (with some complexities such as alternative splicing).
Amplification: Transcription allows for the creation of many identical copies of $RNA$ from a single $DNA$ gene. Subsequently, translation allows for many identical proteins to be made from a single $RNA$ molecule. - This process facilitates a rapid and dramatic response to cellular changes or needs. - Amplification is a luxury, not a necessity; the level of protein production must be matched by the level of regulated gene expression.
Regulation: Both the timing and the amount of $RNA$ produced are strictly regulated during transcription.

Chemical and Structural Differences: DNA vs. RNA

Nucleotide Structure: Both are linear polymers of four nucleotides held together by $5'$ to $3'$ phosphodiester bonds.
Sugar Component: - RNA uses Ribose, which has a hydroxyl ( $-OH$ ) group at the $2'$ position. - DNA uses Deoxyribose, which has a hydrogen ( $-H$ ) at the $2'$ position.
Bases: - RNA contains Adenine ( $A$ ), Guanine ( $G$ ), Cytosine ( $C$ ), and Uracil ( $U$ ). - DNA contains Adenine ( $A$ ), Guanine ( $G$ ), Cytosine ( $C$ ), and Thymine ( $T$ ). - Uracil ( $U$ ) in $RNA$ base-pairs with Adenine ( $A$ ) just as $T$ does in $DNA$ .
Physical Structure: - DNA is typically a double-stranded, stable, and static information store. - RNA is usually single-stranded. This allows for intra-molecular base pairing, resulting in complex $3D$ folding and catalytic properties.

The RNA World Hypothesis

This hypothesis suggests that $RNA$ was the basis of all life before the evolution of $DNA$ and proteins.
Comparitive properties: - DNA: Information storing, stable, static. - RNA: Somewhere in-between; stores information but is unstable; possesses catalytic activity but is inefficient; can be static or dynamic. - Protein: Information-empty, catalytic, dynamic.

Diversity and Function of RNA Molecules

Messenger RNA (mRNA): Comprises approximately $5\%$ of total cellular $RNA$ . It serves as a "middle man" carrying genetic information from the nucleus to the cytosol for translation.
Ribosomal RNA (rRNA): Accounts for approximately $80\%$ of total cellular $RNA$ . These molecules associate with proteins to form ribosomes. - Eukaryotic rRNAs include: $18S, 28S, 5S, \text{ and } 5.8S$ . - Ribosomes possess "peptidyl transferase" activity, which is catalyzed by ribozymes (the RNA itself). - Genes for rRNA are clustered in the Nucleolus.
Transfer RNA (tRNA): The smallest of the three major RNAs. They act as adaptors between $mRNA$ codons and amino acids during protein synthesis.
Micro RNA (miRNA): Single-stranded molecules approximately $21-23$ nucleotides in length. They are transcribed but not translated; they regulate gene expression by binding to and down-regulating $mRNA$ .
Other Small RNAs: Used in processes such as $RNA$ splicing and telomere maintenance.

The Mechanism of Transcription

Unwinding: The process begins with the opening and unwinding of the $DNA$ double helix immediately "upstream" ( $5'$ ) of a gene.
Strand Usage: - Template Strand: The specific $DNA$ strand used as a guide for $RNA$ synthesis. $RNA Polymerase$ reads the template in the $3' \rightarrow 5'$ direction. - Non-template (Coding) Strand: The $DNA$ strand whose sequence matches the newly synthesized $RNA$ (except with $T$ instead of $U$ ).
RNA Polymerase (RNAP): - Catalyzes the formation of phosphodiester bonds between ribonucleoside triphosphates ( $NTPs$ ). - Unlike $DNA Polymerase$ , $RNA Polymerase$ can initiate synthesis without a primer. - The high-energy bonds of the $NTPs$ power the reaction. - Speed: Approximately $30\,\text{bases/second}$ . - Error Rate: $1 \text{ in } 10^4$ . This higher error rate is tolerated because RNAs are short-lived and mutant proteins are transient.
Simultaneous Transcription: Multiple $RNA Polymerases$ can transcribe a single gene simultaneously because the $RNA$ strand does not remain base-paired with the $DNA$ template; the $DNA$ closes back up immediately behind the enzyme.

Transcription in Prokaryotes (Bacteria)

Promoter Recognition: Bacterial $RNA Polymerase$ has a general weak affinity for $DNA$ . It "scans" the helix until it encounters a Promoter sequence.
Sigma (\sigma) Factor: A subunit of the bacterial $RNA Polymerase$ complex responsible for recognizing and binding to the promoter.
Initiation and Termination: - Once initiated, the $\sigma \text{ factor}$ dissociates, allowing the enzyme to continue elongation. - Transcription continues until the polymerase hits a Terminator sequence (stop signal).
Simplicity: In bacteria, newly transcribed mRNAs are immediately bound by ribosomes for translation because there is no nuclear compartment.

Transcription in Eukaryotes

Eucaryotic Complexity: Eukaryotic cells have three types of $RNA Polymerase$ : - RNAP I: Transcribes most $rRNA$ genes. - RNAP II: Transcribes protein-coding genes, $miRNA$ genes, and genes for some small RNAs (e.g., those in spliceosomes). - RNAP III: Transcribes $tRNA$ genes, the $5S rRNA$ gene, and other small RNAs.
General Transcription Factors (GTFs): - Eukaryotic $RNAPs$ cannot initiate transcription alone; they require GTFs to recruit the enzyme, position it, separate the $DNA$ , and launch the polymerase. - TFIID: The first GTF to bind. It contains the TBP (TATA-binding protein) subunit. - TATA Box: A specific promoter sequence rich in $T$ and $A$ (e.g., $TATAAT$ ) found approximately $25$ bases upstream ( $-25$ ) of the start site. - DNA Kinking: Binding of $TBP$ to the TATA box kinks the $DNA$ approximately $90^{\circ}$ , facilitating the assembly of other factors. - Basal Transcription Complex: Includes $TFIIA, TFIIB, TFIID, TFIIE, TFIIF, \text{ and } TFIIH$ .
RNAP II Tail Phosphorylation: - $RNAP II$ has a carboxyl-terminal domain ( $CTD$ ) or "tail." - TFIIH contains a kinase subunit that phosphorylates the tail to trigger the release of the enzyme from the promoter and signal the start of elongation. - Only unphosphorylated $RNAP II$ can be recruited to a promoter; phosphates must be stripped for recycling.

Gene Anatomy and Regulatory Regions

Exons: The protein-coding regions of a gene.
Introns: Noncoding intervening sequences that interrupt exons. They can range from a single nucleotide to $10,000$ nucleotides.
Consensus Sequences: Conserved markers (like the TATA box) found at recognition sites.
Regulatory Regions: - Basal Promoter: Includes the proximal component (TATA box) for positioning and a distal component (e.g., $CAAT$ or $GC$ boxes) that specifies the frequency of initiation. - Enhancers/Repressors: Sequences that regulate expression levels. They can function at great distances (hundreds/thousands of bases) from the start site and are orientation-independent.
Upstream and Downstream: Noncoding regions at the $5'$ end are "upstream"; regions at the $3'$ end are "downstream."

Eukaryotic mRNA Processing

Compartmentalization: Since eukaryotes have a nucleus, mRNAs must be processed and inspected before export to the cytoplasm. This provides Quality Control.
5' Cap: A methylated guanosine residue added in an atypical way to the $5'$ end. It protects from $5'$ exonuclease degradation and helps in ribosome binding.
Poly-A Tail: A sequence of approximately $200-250$ adenine nucleotides added to the $3'$ end by $Poly(A) Polymerase$ . - The Polyadenylation Signal ( $AAUAAA$ ) is recognized by an endonuclease that cleaves the transcript before the tail is added.
Circularization: Interaction between proteins at the $5'$ cap and the $Poly-A$ tail causes the $mRNA$ to circularize, signaling that it is genuine and intact.

RNA Splicing and the Spliceosome

Splicing: The process of removing introns and joining exons together to form a mature, all-coding $mRNA$ .
Spliceosome: A large complex made of five small nuclear RNAs ( $U1, U2, U4, U5, \text{ and } U6$ ) and over $50$ proteins. These are collectively called snRNPs ("snurps").
Mechanism: - Specific sequences at the intron ends (GU at the $5'$ end and AG at the $3'$ end) mark the splice sites. - A "branch point" Adenine ( $A$ ) attacks the $5'$ splice site. - The intron is removed in the shape of a Lariat (a loop structure) and is discarded.
Function of Introns: While often viewed as "junk" or evolutionary relics, introns allow for Alternative Splicing.

Alternative Splicing and Modularization

Alternative Splicing: Splicing non-adjacent exons together to create different protein variants from a single gene. - Exon order is always preserved; introns are always discarded. - Approximately $60\%$ of human genes undergo alternative splicing, greatly increasing "protein potential."
Modular Protein Synthesis: Exons often encode individual functional modules called Domains. Splicing allows these domains to be "traded" or combined like attachments on a kitchen tool (e.g., a mixer with multiple possible attachments like a juicer or pasta roller).

Nuclear Export and mRNA Lifespan

Nuclear Pore Complex (NPC): Highly selective gates. mRNAs are only allowed out if they are bound by specific proteins at the cap, splice junctions, and tail (e.g., Cap-binding protein, Exon Junction Complex (EJC), and Poly-A-binding protein).
Degradation: All RNAs are eventually degraded by RNases. - Lifespan varies from minutes to days. - Lifespan is often controlled by sequences in the 3' UTR (3' Untranslated Region), which lies past the protein-coding sequence. - Inhibition: Regulatory factors (like miRNAs) can bind to the $3' UTR$ to modulate translation or stability.

Clinical Correlation and Pharmacology

Retroviruses (e.g., HIV): Possess an $RNA$ genome and use Reverse Transcriptase to copy $RNA$ into $DNA$ . This is the reverse of the usual information flow. The viral $DNA$ then integrates into the host genome.
Antiviral Drugs: - AZT (Zidovudine) and DdI (Dideoxyinosine): Nucleotide analogues that inhibit reverse transcriptase. Host kinases phosphorylate them, and they are incorporated into viral chains, causing chain termination.
Antibiotics: - Rifampin: Specifically inhibits bacterial $RNA Polymerase$ by interfering with the enzyme-promoter complex. It is a primary treatment for Tuberculosis. - Isoniazid: Often used alongside rifampin as an antimetabolite for tuberculosis treatment.