Molecular Biology: DNA Replication Quiz

DNA Replication, Transcription, and Translation

• This is a study guide for DNA replication, transcription, and translation.
• The key references are Essential Cell Biology by Bruce Alberts, 6th Edition.

DNA Replication

• Replication is a semiconservative process. Each strand of the double helix serves as a template for the new, complementary strand.
• DNA synthesis proceeds only in the 5' → 3' direction.
• Newly added nucleotides are joined by phosphodiester bonds.
• DNA replication is enzymatic, requiring numerous protein complexes.
• Energy is needed for this process, derived from the hydrolysis of high-energy phosphate bonds.
• Key proteins involved:
  • Helicase (MCM 2-7 complex): Unwinds DNA by breaking the hydrogen bonds between complementary nitrogenous bases. The MCM 2-7 proteins use ATP during this process.
  • Topoisomerases: Relieve torsional stress in DNA caused by unwinding. There are two types, topoisomerase I and topoisomerase II.
  • Replication protein A (RPA): Binds to single-stranded DNA segments to prevent the formation of hydrogen bonds and allow DNA polymerase to function.
  • PCNA: A ring-shaped homotrimer that acts as a scaffold for proteins involved in DNA replication, repair, chromatin remodeling, and epigenetic regulation. Protects DNA polymerase from falling off the template strand and releases DNA polymerase after each Okazaki fragment is synthesized.
  • Replication factor C (RFC): Opens PCNA protein trimer in the presence of ATP, guides DNA into the center of the ring, and then closes the ring to aid in PCNA placement.
  • DNA polymerases (α, β, γ, δ, ε): Synthesize DNA; α adds the initial DNA strand; δ and ε are involved in leading and lagging strand synthesis.
  • RNase H: Removes RNA primers except for the last one.
  • FEN1: Removes the last ribonucleotide of the RNA primer.
  • Ligase: Joins deoxyribonucleotides to form phosphodiester bonds by using ATP; specifically, it links Okazaki fragments on the lagging strand.

DNA Replication process limitations/stages

• The need to unwind the DNA double helix.
• The inability of DNA polymerase to independently initiate the synthesis of a new strand.
• The addition of nucleotides occurs only in the 5' → 3' direction.
• The specific starting point and sequence within the DNA strand where replication begins.
• DNA strand replication involves origin recognition and formation of a pre-replication complex (pre-RC).
• The process occurs at origins of replication, which are specific 10,000 base pair sequences in the DNA, thus creating replication factories/foci.
• A fragment of DNA replicated from one origin is called a replicon.
• Replication forks are formed (bidirectional).
• Leading vs. Lagging Strands:
  • The leading strand is synthesized continuously.
  • The lagging strand is synthesized in sections called Okazaki fragments.

Overview of DNA Replication Stages

• Initiation:

  • Recognizing origins of replication.
  • Forming a pre-RC: ORC attachment, recruitment of Cdt1 and Cdc6, and helicase attachment.
  • Separating DNA strands: Helicase breaks hydrogen bonds; Topoisomerases relax supercoils; SSBs stabilize single-stranded DNA to prevent re-annealing.

• Elongation:

  • Leading strand synthesis: Continuous.
  • Lagging strand synthesis: Discontinuous, creating Okazaki fragments.
  • Primers: Synthesized by primase. RNase H/ FEN1 removes primers.
  • Okazaki fragment synthesis: Completed by DNA polymerase.
  • Joining Okazaki fragments: Done by DNA ligase.

• Termination:

  • Meeting of replication forks.
  • Terminal sequences.
  • Disintegration of replication complex.
  • Telomere synthesis.

Transcription

• Transcription is the process of rewriting genetic information from DNA to RNA.
• It is based on the complementarity rule (A-U, C-G).
• The unit of transcription is the section of DNA between the promoter and the terminator.
• DNA is read in the 3' → 5' direction.
• RNA (mRNA, rRNA, tRNA) is synthesized in the 5' → 3' direction.
• The process is catalyzed by RNA polymerases (I, II, and III). Each type synthesizes different kinds of RNA.

Transcription Stages

• Initiation: Formation of a pre-initiation complex involving RNA polymerase and transcription factors. This complex binds to the promoter region to initiate transcription.
• Elongation: RNA polymerase moves along the template strand, synthesizing the RNA transcript in the 5' → 3' direction.
• Termination: Signals from a terminator sequence cause RNA polymerase to detach from the DNA, releasing the newly made RNA molecule.

Post-transcriptional Processing

• Maturation of pre-mRNA to mRNA includes:
  • Capping the 5' end.
  • Splicing: Removing introns and joining exons (RNA splicing).
  • Polyadenylation (adding a poly-A tail) to the 3' end.

Translation

• Translation is the synthesis of a polypeptide chain from an mRNA template.
• This process is catalyzed by ribosomes, taking place in the cytoplasm and on the rough endoplasmic reticulum.
• The core includes polypeptide chain synthesis based on the mRNA sequence and the formation of peptide bonds.

Ribosome Structure

• Ribosomes consist of two subunits: small and large.
• Both subunits contain proteins and rRNA.
• The subunits are only connected during translation.
• Subunits contain binding sites for tRNA (aminoacyl-tRNA (A), peptidyl-tRNA (P), and exit site (E)).

Translation Stages

• Initiation:

  • Pre-initiation complex formation (small ribosomal subunit, initiation factors, and initiator tRNA).
  • Small subunit binds to the mRNA 5'UTR, and scans for an AUG start codon.
  • Large subunit joins, forming a functional ribosome. Initiator tRNA occupies the P site, and the A site is ready for the next aminoacyl-tRNA.

• Elongation:

  • Cycles: Consist of binding of tRNA carrying amino acids at the A site, peptide bond formation in the P site, translocation (shifting of ribosome on mRNA), and release of an uncharged tRNA from the E site.

• Termination:

  • STOP codons (UAA, UAG, UGA) are encountered.
  • Release factors bind to the A site.
  • Peptidyl transferase adds a water molecule to the chain, releasing the completed polypeptide.
  • Ribosome dissociates.

Post-translational Modifications

• Many proteins require modifications to function (correct folding, proteolytic processing, cofactor binding etc.)
• Specific chemical groups may be added or removed (e.g., phosphorylation, glycosylation).
• Chaperone proteins facilitate proper protein folding.
• Incorrectly folded proteins can be degraded.