module 12

Characteristics of Genetic Material and the Central Dogma

• The genetic material of an organism must exhibit four fundamental characteristics:

  1. Replication: The ability to be copied accurately.

  2. Storage of Information: The capacity to hold the complex instructions for an organism.

  3. Expression of Information: This occurs through the processes of transcription and translation.

  4. Variation: The ability to change through mutation and recombination.

• The Flow of Genetic Information: The initial event in this flow is the transcription of DNA, where three main types of RNA molecules are synthesized:

  • Messenger RNA (mRNA): Products of specific genes that encode different proteins.

  • Transfer RNA (tRNA): Mediates the translation of mRNA into proteins.

  • Ribosomal RNA (rRNA): Works alongside tRNA to facilitate protein synthesis.

• The Central Dogma of Molecular Genetics: This principle states that "DNA makes RNA, which makes proteins."

• Translation: This is the process where chemical information in mRNA directs the construction of a chain of amino acids called a polypeptide. This polypeptide then folds into a functional protein.

DNA Replication and the Replication Fork

• Origins and Enzymes:

  • At the origin of replication, topoisomerase II relaxes the supercoiled chromosome.

  • Two replication forks form as helicase opens the double-stranded DNA.

  • Single-stranded binding proteins (SSBs) coat the DNA to keep the strands separated.

• Synthesis Process:

  • Replication occurs in both directions (bidirectional).

  • RNA primase synthesizes an RNA primer complementary to the parental strand.

  • DNA polymerase III elongates this primer by adding nucleotides to the $3'-OH$ end.

  • Leading Strand: DNA is synthesized continuously.

  • Lagging Strand: DNA is synthesized in short stretches called Okazaki fragments.

  • RNA primers on the lagging strand are removed by the exonuclease activity of DNA polymerase I.

  • DNA ligase joins the Okazaki fragments together.

• Building Blocks: The process utilizes deoxyribonucleoside triphosphates ($dATP$, $dTTP$, $dGTP$, $dCTP$), which provide both the necessary nucleotides and the energy required for strand elongation.

Fundamentals of Transcription and the Genetic Code

• Gene Expression Stages:

  • Transcription: Converting DNA into mRNA.

  • Translation: Converting mRNA into protein with the assistance of ribosomes.

• The Genetic Code Features:

  • It is written in linear form using ribonucleotide bases.

  • Triplet Code: Each "word" or codon consists of three ribonucleotide letters.

  • Each triplet specifies only one amino acid, making the code unambiguous.

• DNA Strands in Transcription:

  • Template Strand: The DNA strand read by RNA polymerase ($3' \rightarrow 5'$) to synthesize RNA ($5' \rightarrow 3'$). The resulting mRNA is complementary to this strand.

  • Non-Template Strand (Coding Strand): The DNA strand sequence that is identical to the mRNA sequence (except that T in DNA is replaced by U in RNA). It is not used directly by RNA polymerase but reflects the codons to be translated.

Prokaryotic Transcription in E. coli

• Essential Components:

  1. DNA Template (Gene): The specific strand containing the genetic information.

  2. Promoter Sequence: Specific DNA regions where RNA polymerase binds ($-35$ and $-10$ regions).

  3. RNA Polymerase: The enzyme that synthesizes RNA.

  4. Sigma Factor ($\sigma$): Helps the polymerase recognize and bind to the promoter.

  5. Ribonucleoside Triphosphates (NTPs): The building blocks of RNA ($ATP$, $UTP$, $GTP$, $CTP$).

  6. Termination Signals: DNA sequences signaling the end of transcription.

• Transcription Site Numbering:

  • $+1$ Site (Initiation Site): The nucleotide pair where the first $5'$ mRNA nucleotide is transcribed.

  • Upstream Nucleotides: Designated with negative numbers ($-$) and precede the initiation site.

  • Downstream Nucleotides: Designated with positive numbers ($+$) and follow the initiation site.

• The Promoter: A sequence upstream of the gene where RNA polymerase binds. Its specific sequence determines the frequency of transcription.

  • $-10$ Region (Pribnow Box): Consensus sequence is $TATAAT$.

  • $-35$ Region: Consensus sequence is $TTGACA$.

  • These regions are often AT-rich to facilitate DNA unwinding.

RNA Polymerase and Bacterial Chromosome Structure

• RNA Polymerase Holoenzyme: In E. coli, one enzyme transcribes all genes. It consists of five subunits:

  • Core Enzyme: Two $\alpha$ subunits (polymerase assembly), one $\beta$ subunit (binds incoming NTPs), and one $\beta'$ subunit (binds the DNA template).

  • Sigma ($\sigma$) Factor: Functions only during initiation to ensure correct promoter recognition. When combined with the core enzyme, it forms the holoenzyme.

• Bacterial DNA: Circular, double-stranded, and compacted into a nucleoid. It lacks a membrane-enclosed nucleus.

  • Supercoiling: Facilitated by topoisomerases, which cut, unwind, and rejoin strands to compact the DNA. Supercoiled DNA is more compact and sediments more rapidly than linear forms.

• Genomic Organization:

  • Prokaryotic genomes are compact and often polycistronic, meaning one mRNA molecule contains multiple coding regions (cistrons).

  • Operon: A group of genes transcribed together under one promoter (e.g., the lac operon containing lacZ, lacY, and lacA).

  • Transcription, translation, and mRNA degradation occur simultaneously because there is no nucleus.

Steps of Prokaryotic Transcription

1. Initiation: The RNA polymerase holoenzyme binds to the $-35$ and $-10$ sequences and unwinds the DNA locally.

2. Promoter Clearance / Start of RNA Synthesis: RNA polymerase starts synthesizing the strand. The sigma factor dissociates after the first few nucleotides are added.

3. Elongation: RNA polymerase moves $3' \rightarrow 5'$ along the template, synthesizing RNA in the $5' \rightarrow 3'$ direction.

4. Termination: Occurs via two mechanisms:

   • Rho-dependent termination: Controlled by the rho ($\rho$) protein, a hexamer helicase. Rho moves along the mRNA and, when the polymerase stalls at a G-rich region, rho catches up to release the mRNA.

   • Rho-independent termination: Initiated by a specific DNA sequence that creates a GC-rich hairpin loop followed by a U-rich region in the mRNA. This stalls the polymerase and allows the weak RNA-DNA interaction to break.

Eukaryotic Transcription Mechanisms

• Comparison with Prokaryotes:

  • Eukaryotes have a membrane-bound nucleus; mRNA must be transported to the cytoplasm.

  • Eukaryotic mRNA is generally monocistronic (one mRNA codes for one protein).

  • Eukaryotes use three different RNA polymerases.

• Initiation:

  • Requires Basal Transcription Factors ($TFIIA$, $TFIIB$, $TFIID$, etc.) to recruit the polymerase.

  • TFIID contains the TATA-binding protein (TBP), which binds to the TATA Box (Goldberg-Hogness Box).

  • The TATA Box is located at approximately $-25$ to $-30$ upstream of the $+1$ site, with the consensus sequence $TATAAA$.

• Additional Promoter Elements:

  • CAAT Box: Located around $-80$ upstream.

  • GC-rich and Octamer Boxes: Found further upstream in highly active genes to bind regulatory proteins.

  • Regulatory Elements: Enhancers (increase transcription efficiency) and Silencers (decrease it).

• Elongation and Chromatin:

  • DNA is wrapped around histones in nucleosomes. The FACT complex (Facilitates Chromatin Transcription) removes histones temporarily for the polymerase and replaces them afterward.

• Termination by Polymerase Type:

  • RNA Polymerase I: Uses a specific $18-nucleotide$ termination sequence.

  • RNA Polymerase II: Continues $1,000$ to $2,000$ nucleotides past the gene; the tail is removed during processing.

  • RNA Polymerase III: Forms an RNA hairpin similar to rho-independent termination.

Eukaryotic mRNA Processing

• Post-transcriptional Modification: Pre-mRNA must be processed to increase stability and lifespan.

  1. $5'$ Capping: Addition of a $7-methylguanosine$ cap. It protects against degradation and helps ribosomes recognize the mRNA.

  2. $3'$ Poly-A Tail Addition: The pre-mRNA is cleaved near the $AAUAAA$ signal, and Poly-A polymerase adds approximately $200$ adenine nucleotides. This assists in nuclear export and stability.

  3. RNA Splicing: Removal of introns (non-coding) and joining of exons (coding).

• Spliceosome Machinery: A ribonucleoprotein (RNP) complex composed of five small nuclear ribonucleoproteins (snRNPs U1, U2, U4, U5, and U6) and hundreds of proteins.

  • $5'$ Splice Site (Donor Site): Starts with the consensus sequence $GU$.

  • $3'$ Splice Site (Acceptor Site): Ends with the consensus sequence $AG$.

  • Branch Point Sequence: Contains a conserved adenine (A) used for lariat formation.

  • Splicing Catalysis: The branch point A attacks the $5'$ end of the intron, forming a $2' \rightarrow 5'$ bond and a lariat loop. The $3'-OH$ of the $5'$ exon then attacks the $3'$ splice site to ligate exons.

Alternative Splicing and RNA Editing

• Alternative Splicing: Allows one gene to produce multiple proteins with different functions, generating transcriptome diversity.

  • Example: The Dscam gene in Drosophila melanogaster has $116$ exons and can produce over $18,000$ different proteins.

• RNA Editing: Post-transcriptional modification of nucleotides.

  1. Deamination of Cytidine: Performed by APOBEC-1, resulting in uridine ($U$).

  2. Deamination of Adenosine: Performed by ADAR, resulting in inosine (I). Inosine behaves like guanosine ($G$) during base pairing.

• ORF (Open Reading Frame): A continuous stretch of RNA starting with an initiation codon (usually $AUG$) and ending with a stop codon ($TAA$, $TAG$, or $TGA$).

Translation Machinery and tRNA

• Ribosomes: Large and small subunits combined. Prokaryotic ribosomes are $70S$; Eukaryotic ribosomes are $80S$.

• tRNA (Transfer RNA):

  • Small, stable molecules ($75-90$ nucleotides) with a cloverleaf structure (four stems, three loops).

  • The anticodon loop complements the mRNA codon.

  • The amino acid is covalently linked to the CCA end.

• Aminoacyl-tRNA Synthetases: Enzymes that "charge" tRNAs with the correct amino acid using $ATP$.

• Ribosomal Binding Sites:

  • A (Acceptor) Site: Where charged tRNAs enter.

  • P (Peptidyl) Site: Holds the tRNA with the growing polypeptide chain.

  • E (Exit) Site: Where empty tRNAs leave the ribosome.

Important Logistics

• Midterm 2: Covers specific lectures.

• Final Exam: April 22, 2026, at $2:00\,PM$. It is cumulative, and a grade of $35\%$ is required to pass the course.