Molecular Biology: DNA Replication & Translation

DNA Replication, Transcription, and Translation

• This is a summary of the processes of DNA replication, transcription and translation.
• The lectures will focus on these processes as outlined in Essential Cell Biology, 6th Edition by Bruce Alberts.
• Key chapters are 6: DNA Replication and Repair and 7: From DNA to Protein.

DNA Replication

• Replication is a semiconservative process, meaning each strand of the double helix acts as a template for a new, complementary strand.
• DNA synthesis proceeds only in the 5' to 3' direction.
• Newly added nucleotides are connected by a phosphodiester bond.
• The process is enzymatic due to the numerous protein complexes required.
• Energy input is necessary, supplied by the hydrolysis of high-energy phosphate bonds.

Replication Proteins

• Helicase (MCM 2-7): A protein complex forming a ring around a single DNA strand; it breaks hydrogen bonds between complementary base pairs, unwinding the DNA helix. The MCM 2-7 proteins use ATP hydrolysis to move along the strand and separate the complementary strands.
• Topoisomerases: Enzymes that relieve torsional stress (twisting tension) in DNA during unwinding. Topoisomerases change the topology of the DNA molecule, reducing tension. There are two main types: Topoisomerase I and II.
• Replication Protein A (RPA): Binds to single-stranded DNA to prevent the formation of secondary structures, making the DNA available for replication.
• Proliferating Cell Nuclear Antigen (PCNA): A ring-shaped homotrimer that acts as a scaffold for proteins involved in replication and repair, chromatin remodeling, and epigenetic regulation. It protects DNA polymerase from falling off the template strand and releases it after each Okazaki fragment is synthesized.
• Replication Factor C (RFC): A ring-applying protein required for loading PCNA onto the DNA strand during replication. The process depends on ATP in both the leading and lagging strand.
• DNA Polymerases: Enzymes responsible for synthesizing new DNA strands using existing DNA as templates. Specific types such as alpha (α), beta(β), gamma (γ), delta (δ), and epsilon (ε) have differing roles, which will be covered in detail during the lectures.
• RNase H (endonucleases): Removes RNA primers except for the final ribonucleotide in the newly formed DNA chain.
• FEN1 (exonuclease): Removes the final ribonucleotide of the RNA primer.
• Ligase: Seals the gaps (nicks) between Okazaki fragments on the lagging strand, connecting them using a phosphodiester bond, and using ATP as energy source.

DNA Polymerase

• DNA polymerases need a DNA template for synthesis.
• They cannot initiate synthesis without a primer.
• DNA polymerases have a 5' → 3' polymerase activity and a 3' → 5' exonuclease activity (proofreading).

Polymerase α/primase

• Plays a combined primase and DNA polymerase role.
• Primase synthesizes RNA primers, short strands of RNA complementary to the DNA template.
• Polymerase α adds a short section of DNA (initiator DNA) to these primers.

Ribonucleases

• Enzymes that divide RNA molecules into shorter chains or individual nucleotides, by hydrolyzing phosphodiester bonds.
• Endo-RNases cut RNA molecules in the middle.
• Exo-RNases remove nucleotides from either ends.

DNA Replication Limitations

• Need to unwind the double helix.
• DNA polymerase's inability to initiate synthesis independently.
• Addition of nucleotides can only occur in the 5′ -> 3′ direction.

DNA Replication Stages

• Initiation: Unwinding the DNA helix at origins of replication which involve specific sequences of ~10,000 base pairs. Replication occurs simultaneously at many sites. Clusters occur in what are called replication factories, or foci.
• Elongation: Process of replication forks and copying of parent DNA strands.
• Termination: Completion and assembly of new DNA strands once replication forks meet.

Limitations of Replication

• Primer synthesis for initiating new DNA strands, by primase. Polymerase a synthesizes short strands of 10 nucleotides of iRNA. Polymerase a synthesizes the first ~20-30 deoxynucleotides of DNA (IDNA). Polymerase δ or ε takes over.
• The need to unwind the double helix by helicase
• The inability of DNA polymerase to independently initiate the synthesis of a new DNA strand.
• Addition of nucleotides occurs in a 5' → 3' direction.

Replication Forks

• Due to the antiparallel nature of DNA strands and the synthesis direction, replication forks are asymmetric.
• Leading strand synthesizes continuously.
• Lagging strand synthesizes in short sections, Okazaki fragments.

DNA Replication Elongation (Leading Strand)

• Hydrolysis of one phosphate bond from dNTP provides energy for attachment.
• Formation of a phosphodiester bond between the last deoxyribonucleotide of DNA and 5′ end of new deoxynucleotide.
• Release of pyrophosphate (PPi) makes the reaction irreversible.

DNA Replication Elongation (Lagging Strand)

• Elongation is discontinuous: iRNA synthesis for primer strands.
• Synthesis of subsequent iDNA strands in 5' → 3' direction using a-polymerase.
• Recognition of the primer-template hybrid by RFC, which places PCNA at the site.
• Exchange of a-polymerase for ε-polymerase.

Okazaki fragments

• After reaching the preceding primer, the first a-polymerase with primase detaches from the template.
• DNA strand synthesis in the 5' → 3' direction is catalyzed by ε-polymerase.
• Removal of the iRNA and iDNA fragments using RNase H and FEN1.
• Exchange of ε-polymerase for α-polymerase.

Overview

• The lagging strand DNA synthesis requires multiple primers which are eventually removed.
• In linear DNA molecules, the lagging strand is always shorter than the template, which occurs at the end of each chromosome, the telomeric part.

Post-transcriptional Modifications

• Adding a cap to the 5' end.
• Cutting out introns and joining exons (splicing).
• Adding a series of adenine nucleotides to the 3' end (polyadenylation).

Transcription

• Rewriting genetic information from DNA to RNA.
• Based on the complementarity rule: A-U, C-G.
• DNA strand read in 3' → 5' direction.
• RNA synthesized in 5' → 3' direction.
• Catalyzed by RNA polymerases (I, II, III).
• Occurs in the nucleus in eukaryotes

Initiation of mRNA Transcription

• A complex of proteins recognizes the promoter and creates a platform.
• Transcription factors (one of which is helicase) bind to the platform.
• RNA polymerase II along with transcription factors form the pre-initiation complex that binds to the gene promoter.
• This allows RNA polymerase II to begin transcription of pre-mRNA.

Elongation of mRNA Transcription

• Once elongation begins, most transcription factors detach.
• Phosphate groups are added to the tail of RNA polymerase II, freeing it from the transcription factors.
• More ribonucleotides are added, creating pre-mRNA on the template strand.

Termination of mRNA Transcription

• RNA polymerase II continues transcription until signal to stop.
• Transcription terminator is a DNA fragment marking the end of the gene.
• The steps are:
• phosphodiester bond formation ceases.
• Pre-mRNA dissociates from the DNA-RNA hybrid.
• The unravelled DNA fragment joins to complete the termination.

Translation

• Synthesis of a polypeptide chain from mRNA.
• Occurs in the cytoplasm or on membranes of the rough endoplasmic reticulum.
• Catalyzed by ribosomes.

Ribosome Structure

• Ribosomes are made up of two subunits, small and large.
• These subunits are made of proteins and rRNA (ribosomal RNA).
• The small and large subunits are joined during translation.
• mRNA binding site is in the small subunit only.
• Both subunits participate in tRNA binding sites: A, P, E.

Translation Stages

• Initiation: Formation of a pre-initiation complex (small ribosomal subunit, initiation factors, initiator tRNA).
• Elongation: Cycle with 4 stages:

1. tRNA carrying the next amino acid binds to free A site
2. Peptide bond is formed (peptidyl transferase).
3. tRNA without amino acid moves to site E, peptidyl-tRNA moves from site A to site P.
4. Small subunit moves 3 nucleotides, tRNA without amino acid is removed, site A released.

• Termination: Signaled by a STOP codon; factors detach, the polypeptide chain detaches, and the ribosome disintegrates into its subunits.

Post-Translational Modifications

• Many proteins must undergo further modifications to become fully functional.
• Proteolytic processing, formation of secondary structure, protein folding, and various other modifications are included.
• So-called chaperone proteins help in the correct folding, degradation of incorrectly folded proteins.
• Chemical processes can also modify proteins: phosphorylation, dephosphorylation, glycosylation, hydroxylation and methylation.