Transcription and Gene Expression Mechanism and the Mechanisms of Gene Expression

Overview of Gene Expression and Central Dogma

• Gene expression is the biological process of converting genetic information stored in DNA into functional proteins.
• This process is divided into two primary stages:
  • Transcription: The conversion of DNA into messenger RNA ($mRNA$). This is the focus of Chapter 3.
  • Translation: The conversion of $mRNA$ into a chain of amino acids, which ultimately forms a protein. This is the focus of Chapter 4.
• The cell is the basic unit of living tissue. Within human cells, the nucleus contains the genome, split among $23$ pairs of chromosomes.
• Each chromosome consists of a long strand of DNA tightly packaged around proteins known as histones. Specific sections of this DNA are called genes, which contain instructions for making proteins.

The Process of Transcription

• Transcription is the first stage of gene expression.
• When a gene is "switched on," an enzyme called RNA polymerase attaches to the start of the gene.
• RNA polymerase moves along the DNA, unzipping the double helix and reading one of the two strands (the template strand).
• It uses free bases in the nucleus to assemble a strand of $mRNA$.
• The DNA code determines the order of the bases added to the $mRNA$. In RNA, the base Thymine ($T$) is replaced by Uracil ($U$), which is a close chemical cousin.
• Transcription Unit: A region of DNA extending from the promoter to the terminator.
• Coding Strand vs. Template Strand:
  • Template Strand: The strand used by RNA polymerase to make the complementary $mRNA$.
  • Coding Strand: The DNA strand whose sequence matches the resulting $mRNA$ (with $U$ replacing $T$).

Experimental Confirmation of messenger RNA

• Scientists sought to determine how information moved from the nucleus (DNA site) to the cytoplasm (protein synthesis site).
• An experiment was performed using $E. coli$ bacteria:
  • Bacteria were grown for several generations on media containing heavy isotopes: Nitrogen-15 ($^{15}N$) and Carbon-13 ($^{13}C$).
  • All cellular components, including ribosomes, became "heavy labeled."
  • The bacteria were then infected with the T2 bacteriophage virus.
  • The phage destroyed the bacterial DNA and substituted its own genetic information to direct viral protein synthesis.
• Hypothesis: If ribosomes carried specific gene information, new virus-specific ribosomes would need to be made. If ribosomes were passive sites, old bacterial ribosomes could be used to make viral proteins.
• Results: Radio-labeled phage RNA was found associated with all the old bacterial ribosomes. This confirmed that ribosomes do not carry the genetic information themselves; rather, an unstable class of RNA (messenger RNA) carries the code from DNA to the ribosome.

Molecular Components of Transcription

• Transcription requires an enzyme (RNA polymerase) to catalyze the formation of phosphodiester bonds.
• Chemistry of Synthesis:
  • The hydroxyl ($OH$) group attacks the alpha phosphate of an incoming nucleotide, removing a pyrophosphate.
  • Requirements: A DNA template, and the nucleotides ATP, CTP, GTP, and UTP.
  • Direction: The RNA chain grows in the $5'$ to $3'$ direction.
  • Unlike DNA polymerase, RNA polymerase does not require an RNA primer to initiate synthesis.
• Prokaryotic RNA Polymerase Subunits:
  • Core Enzyme: Composed of two $\alpha$ subunits, one $\beta$ subunit, one $\beta'$ subunit, and one $\omega$ subunit.
  • Holoenzyme: The core enzyme plus the $\sigma$ (sigma) factor. Only the holoenzyme can initiate transcription.
  • $\alpha$ Subunits: Encoded by $RPOA$; helps recognize the UP element in strong promoters.
  • $\beta$ and $\beta'$ Subunits: Encoded by $RPOB$ and $RPOC$; involved in DNA binding and phosphodiester bond formation.
  • $\sigma$ Factor: Encoded by $RPOD$; directs the enzyme to the promoter and provides specificity. It dissociates from the core enzyme after initiation and can be recycled.

Promoter Sequences and Numbering

• Nucleotide Numbering:
  • The first nucleotide at the transcription start site is numbered $+1$.
  • Downstream sequences move in the positive direction ($+2$, $+3$, etc.).
  • Upstream sequences (before the start site) are numbered negatively ($-1$, $-2$, etc.).
• Prokaryotic Promoters:
  • Consists of two critical consensus sequences: the $-10$ TATA sequence and the $-35$ sequence.
  • Strong promoters may include an "UP element" located between $-40$ and $-60$.
• Eukaryotic Promoters:
  • Primarily involves RNA Polymerase II for $mRNA$.
  • A key consensus sequence is the TATA box located at approximately $-25$.

Stages of Transcription: Initiation, Elongation, and Termination

• Initiation:
  • The $\sigma$ factor directs the holoenzyme to the promoter to form a "closed promoter complex."
  • The holoenzyme melts a short region of DNA ($10$ to $17$ base pairs) to create an "open promoter complex," also called a transcription bubble.
  • The first base in the RNA is typically a purine ($A$ or $G$).
• Elongation:
  • Once the first few phosphodiester bonds are formed, the $\sigma$ factor dissociates.
  • The core enzyme continues adding nucleotides sequentially.
  • The transcription bubble consists of approximately $14$ unpaired bases; the first $9$ are used to transcribe new RNA.
  • Physical Topology: Opening the helix introduces positive supercoils ahead of the polymerase and negative supercoils behind it. Topoisomerase is required to unwind positive supercoils.
  • RNA polymerase may pause or backtrack to proofread the newly synthesized RNA.
  • Transcription Bubble Detection: Dimethyl sulfate ($DMS$) transfers a methyl group to Adenine in open regions. S1 nuclease then cleaves the un-base-paired DNA, which can be visualized on a gel to identify the bubble's location (e.g., between $-9$ and $+3$).
• Termination:
  • Intrinsic (Rho-independent) Terminator: Involves a GC-rich inverted repeat that forms a hairpin loop, followed by a string of $7$ to $9$ Uracil ($U$) bases. This structure causes the polymerase to fall off.
  • Rho-dependent Terminator: Lacks the poly-U string. A protein factor called Rho follows the RNA polymerase and catches it when it pauses at a hairpin, unwinding the RNA-DNA hybrid to release the transcript.

Regulation of Bacterial Transcription

• Gene expression is energetically expensive, so cells regulate which genes are active. $E. coli$ has more than $3,000$ genes, but not all are transcribed simultaneously.
• Strategies for Regulation:
  1. Alternative Sigma Factors: Viruses like $SpO1$ use specific proteins ($gp28$, $gp33$, $gp34$) to redirect host RNA polymerase to viral genes. Similarly, $E. coli$ uses $\sigma^{32}$ for the heat shock response.
  2. Anti-sigma Factors: The $RSD$ protein binds to $\sigma^{70}$ during environmental stress, blocking its activity.
  3. RNA Polymerase Switching: Bacteriophage T7 encodes its own RNA polymerase to transcribe its late-phase genes.
  4. Anti-termination: Proteins like the N and Q proteins in lambda phage prevent premature termination, allowing read-through into subsequent genes.
  5. Operons: Groups of contiguous, coordinate-controlled genes transcribed as a single "polycistronic" message.
  6. Transcription Attenuation: Premature termination based on leader sequences (e.g., the $trp$ operon).
  7. Riboswitches: RNA regions in the $5'$ untranslated region ($UTR$) that change conformation upon binding a ligand (e.g., FMN binding in the $ribD$ operon).

The Operon Model and Positive/Negative Control

• Negative Control (Induction): A repressor binds a promoter to inhibit transcription until an inducer is present (e.g., Lac operon).
• Negative Control (Repression): An inactive repressor becomes active only in the presence of a co-repressor (e.g., Tryptophan operon).
• Positive Control: An activator (like the CAP-cAMP complex) must bind to the promoter for full transcription activity.
• The Lac Operon:
  • In the absence of lactose, the $lacI$ gene produces a repressor that binds the operator, blocking transcription.
  • In the presence of lactose, the inducer binds the repressor, causing it to release the operator.
  • Catabolite Repression: If glucose is present, cAMP levels are low. If glucose is absent, cAMP levels rise, forming the CAP-cAMP complex, which acts as an enhancer for the lac operon.
  • The lac operon is fully active only when glucose is absent and lactose is present.

Eukaryotic Transcription Machinery

• Eukaryotes use three distinct RNA polymerases:
  • RNA Polymerase I: Located in the nucleolus; synthesizes large ribosomal RNA ($rRNA$) precursors ($28S$, $18S$, $5.8S$).
  • RNA Polymerase II: Located in the nucleoplasm; synthesizes $mRNA$ precursors.
  • RNA Polymerase III: Located in the nucleoplasm; synthesizes $tRNA$ precursors, $5S$ $rRNA$, and small nuclear RNAs.
• Transcription Factors ($TF$):
  • General Transcription Factors (GTFs): Required for basal level transcription. For Pol II, the assembly sequence is: $TF2D$ (containing TATA-binding protein or $TBP$) + $TF2A$ \u2192 $TF2B$ \u2192 $TF2F$/Polymerase II \u2192 $TF2E$ \u2192 $TF2H$.
  • Specific Transcription Factors (Activators): Bind to enhancers and contain DNA-binding motifs.
• DNA-Binding Motifs:
  • Zinc-containing modules: e.g., Zinc finger (coordinated by two Cys and two His) or the $GAL4$ bimetal cluster (six Cys at a zinc ion).
  • Homeodomain: Contains $60$ amino acids and a helix-turn-helix motif.
  • BZIP and BHLH: Dimerize via a leucine zipper.

Epigenetic Regulation in Eukaryotes

• DNA is packaged into nucleosomes: $147$ base pairs of DNA wrapped in $1.75$ turns around an octamer of core histones ($H2A$, $H2B$, $H3$, and $H4$).
• Histone Modification: Covalent changes (acetylation, methylation, phosphorylation) to positively charged histone tails alter their interaction with negatively charged DNA, making DNA more accessible to RNA polymerase.
• Chromatin Remodeling: Complexes use energy to move nucleosomes so RNA polymerase can access genes.
• Overcoming the Nucleosomal Barrier: During elongation, polymerase moves through nucleosomes via nucleosome mobilization (octamer transfer) or $H2A/H2B$ dimer depletion.
• Heterochromatin vs. Euchromatin:
  • Euchromatin: Open, accessible, and transcriptionally active.
  • Heterochromatin: Tightly bundled, repressive, and methylated (often at CpG islands).

Post-Transcriptional mRNA Processing

• Eukaryotic genes contain exons (coding) and introns (intervening sequences).
• Three primary modifications occur after transcription:
  1. Five' Cap ($5'$ cap):
  • Step 1: RNA triphosphatase removes one phosphate.
  • Step 2: RNA guanine transferase adds a GMP molecule.
  • Step 3: Guanine N7 methyltransferase adds a methyl group to the added guanine.
  • Function: Protects RNA and serves as a binding site for ribosomes.
  1. Polyadenylation ($3'$ tail):
  • Cleavage occurs after a $CA$ dinucleotide between the $AAUAAA$ hexamer and a downstream GU-rich region.
  • Poly A polymerase adds approximately $200$ Adenosine ($A$) residues to the site.
  1. Splicing:
  • Carried out by the spliceosome (comprising snRNPs $U1$, $U2$, $U4$, $U5$, and $U6$).
  • Introns typically start with $GU$ (at the $5'$ splice site) and end with $AG$ (at the $3'$ splice site).
  • Mechanism: Two-step transesterification forms a lariat structure from the intron, which is then released and degraded.
  • Alternative splicing allows a single gene to produce multiple protein variants.

Non-Coding RNAs and RNA Interference ($RNAi$)

• Approximately $98$% of the transcriptional output of the human genome is non-coding RNA.
• Small Non-coding RNAs:
  • microRNA ($miRNA$): Endogenous; cut from larger hairpin precursors by the enzyme Dicer into approximately $22$ nucleotides. They often bind imperfectly to targets to inhibit translation.
  • small interfering RNA ($siRNA$): From exogenous sources (viruses or synthetic). Cut by Dicer into $21$ to $25$ nucleotides. They match targets perfectly, leading to mRNA cleavage.
• Mechanism of Action:
  • Dicer cuts the RNA, which is then passed to the RNA-induced silencing complex ($RISC$).
  • The Argonaut protein in the $RISC$ complex guides the antisense strand to the target $mRNA$.
  • $RITS$ (RNA-induced initiation of transcriptional silencing) complexes can also recruit chromatin remodeling enzymes to methylate DNA and histones, resulting in transcriptional silencing.