Central Dogma and DNA Replication Quiz

Central Dogma

• The central dogma describes the flow of genetic information, from DNA to RNA to protein.
• DNA is transcribed into RNA.
• RNA is translated into protein.

Learning Outcomes

• Describe DNA replication, comparing leading and lagging strands.
• Describe the process of transcription.
• Describe the process of translation.
• Gain an overview of post-translational modifications.

DNA Replication

• DNA replication produces two identical DNA molecules, ensuring the transmission of genetic information faithfully.
• Replication occurs at origins of replication.
• Prokaryotic DNA typically has one origin.
• Eukaryotic DNA has multiple origins.
• Replication occurs during the S phase of the cell cycle.
• Replication is bidirectional - originating from multiple origins
• Uses a variety of proteins including DNA polymerase, helicase, and single-strand binding proteins to unwind and copy DNA
• DNA polymerase is a hand-shaped enzyme that copies DNA
• Leading strand synthesized continuously.
• Lagging strand synthesized in short fragments (Okazaki fragments)
• Enzymes (RNase H & DNA ligase) work together to link Okazaki fragments
• DNA polymerase moves along template strands in 3' → 5'.

Cell Cycle (Mitosis)

• Nucleic acids store and transmit genetic information.
• Before cell division, DNA replicates with high fidelity.
• The cell cycle progresses through G0, G1, S, G2, and M phases.

Origin of Replication (ori)

• Origin of replication (ori) is a specific region of the DNA sequence in the genome where DNA replication is first initiated.
• Prokaryotes usually have one origin of replication.
• Eukaryotes often have multiple origins for efficiency.
• The origins of replication are activated in the S phase of the cell cycle.

Transcription

• Transcription is the process of making an RNA copy of a DNA sequence.
• Prokaryotic transcription occurs in the cytosol
• Eukaryotic transcription occurs within the nucleus.
• Transcription is initiated at specific regions called promoters.
• Requires RNA polymerase.
• 3 stages: initiation, elongation, and termination.
• Prokaryotes have one type of RNA polymerase.
• Eukaryotes have 3 different RNA polymerases. RNA polymerase II primarily controls mRNA synthesis.

Transcription in Prokaryotes

• RNA polymerase binds to the promoter region.
• RNA polymerase unwinds a section (transcription bubble) and synthesizes an RNA copy using NTPs.
• RNA copy is synthesized 5' → 3' antiparallel and complementary to the template strand which is 3' → 5'
• Termination is through Rho-dependent or Rho-independent mechanisms, which results in RNA termination and separation from the template DNA.

Promoter

• Promoter is the starting point of transcription.
• Consensus sequences are recognized by RNA polymerase.
• Includes -35 and -10 regions (prokaryotic)
• TATA box is a consensus sequence for eukaryotic initiation.
• Upstream elements/activation sequences also found on eukaryotic genes

Elongation (prokaryotes)

• RNA polymerase moves along the anti-sense strand.
• NTPs complementary to the template are added.
• Phosphodiester bonds form between the nucleotides.
• DNA unwinds to expose the template strand for continuing elongation.
• Topoisomerases are enzymes which relieve supercoiling stress during transcription

Termination (prokaryotes)

• Rho-independent termination: hairpin structure followed by a U-rich region. The hairpin causes RNA polymerase to pause.
• Rho-dependent termination: Rho factor binds to RNA and moves towards RNA polymerase, causing it to release from the DNA

Transcription in Eukaryotes

• More complex than prokaryotic transcription.
• Occur in the nucleus.
• Multiple RNA polymerases (RNA polymerase II specifically for mRNA synthesis)
• mRNA is synthesized in 5' → 3' direction.
• Eukaryotic forms have TATA box, Inr, Upstream elements in the promoter regions.

Initiation (eukaryotes)

• TBP binds to the TATA box of the promoter, initiating the unwinding of the DNA.
• Transcription factors (TFIIB and TFIIA) bind, along with RNA polymerase II.
• Other factors bind to form a complete pre-initiation complex.

Other Eukaryotic Transcription Factors

• Activator proteins bind to enhancers which are sequences further away from the promoter regions
• Coactivators interact with the pre-initiation complex to enhance transcription rate.
• Repressors bind to proteins to suppress transcription rate

mRNA produced in prokaryotes and eukaryotes

• Prokaryotic mRNA is polycistronic (codes for multiple proteins).
• Eukaryotic mRNA is monocistronic (codes for a single protein)
• Eukaryotic mRNA undergoes extensive processing. (removal of introns, 5' cap, 3' polyA tail.)

Post-transcriptional processing in eukaryotes

• Capping (5' end).
• Addition of a poly(A) tail (3' end).
• Removal of introns (splicing) and joining of exons (splicing).
• This processing produces the mature mRNA used for translation

Translation

• Ribosomes translate mRNA into polypeptide chains, directing amino acids in based on the order in the mRNA.
• Uses mRNA, tRNA, and ribosomes.
• Genetic code is degenerate, meaning one amino acid is coded by multiple codons. The wobble hypothesis describes flexibility with the third position of the codon.
• mRNA is read 5′ to 3′ and protein synthesis occurs from amino to carboxyl
• Three stages: Initiation, Elongation, Termination

Translation (Initiation- Eukaryotes)

• Small ribosomal subunit binds to the mRNA 5' cap.
• Initiator tRNA (Met-tRNA) binds, along with various initiation factors.
• Large ribosomal subunit joins the complex to form the complete ribosome.
• Ribosome moves to the start codon (AUG).

Translation (Initiation- Prokaryotes)

• Small ribosomal subunit binds to Shine-Dalgarno sequence
• Initiator tRNA (Met-tRNA) binds
• Large subunit joins
• Ribosome identifies start codon (AUG)

Translation (Elongation)

• Aminoacyl-tRNA synthetases are enzymes that attach amino acids to tRNAs.
• Incoming aminoacyl-tRNA binds at the A site.
• Peptide bond forms between the amino acids.
• Ribosome moves down the mRNA.
• Empty tRNA moves to E-site and is released.

Translation (Termination)

• Stop codon (UAA, UAG, or UGA) enters the A-site.
• No tRNA matches the stop codon.
• Release factors bind, causing the polypeptide chain to be released and the ribosome to dissociate.

Post-translational modifications

• Occur in eukaryotes.
• Modifications include glycosylation, phosphorylation, and others.
• These modifications play crucial roles in protein function and activity

DNA Topoisomerases

• Enzymes that relieve supercoiling stress in DNA during replication and transcription.
• Essential in regulating the topological state of the genetic material.