DNA Replication: Enzymes and Process

DNA Replication Overview

• DNA replication is how cells duplicate DNA before dividing
• DNA replication is semi-conservative
• Each new DNA molecule has one old (parental) strand and one newly synthesized strand
• Genetic material must facilitate replication, information storage, expression, and mutation variation
• DNA consists of deoxyribose, phosphate groups, and nitrogenous bases

Key Enzymes in DNA Replication

• Helicase unwinds the double-stranded DNA helix at the replication fork, separating parental strands and creating single-stranded DNA templates
• Single-Stranded Binding Proteins (SSBs) bind to single-stranded DNA, preventing re-annealing or degradation
• Topoisomerase (Gyrase) relieves torsional strain from helicase activity, making temporary DNA cuts to release tension before re-ligating strands
• Primase synthesizes short RNA primers (10–12 nucleotides) for DNA polymerase initiation
• DNA Polymerase III is the primary enzyme that adds nucleotides to the growing DNA strand during replication and synthesizes the new strand in a 5' → 3' direction
• DNA Polymerase I removes RNA primers laid down by primase and replaces them with DNA nucleotides, using exonuclease activity to remove RNA primers and repair DNA
• Ligase seals "nicks" in the sugar-phosphate backbone, joining Okazaki fragments on the lagging strand of DNA
• DNA Polymerase II is involved in DNA repair and helps correct mistakes made during replication

Key Steps in DNA Replication

• Initiation starts when initiator proteins recognize and bind to the origin, beginning the unwinding process
• Helicase unwinds DNA, with single-strand binding proteins stabilizing separated strands
• Primase synthesizes RNA primers, which allows DNA polymerase to start synthesis
• Elongation begins as DNA polymerase III synthesizes the new DNA strand in a 5' → 3' direction by adding nucleotides
• The leading strand is synthesized continuously, whereas the lagging strand is synthesized in short fragments (Okazaki fragments)
• DNA polymerase I replaces RNA primers with DNA, and ligase seals any remaining gaps
• Termination occurs when two replication forks meet or when a termination sequence is reached, where topoisomerase prevents tangling

Transcription Overview (DNA to RNA)

• Transcription is the process by which an RNA molecule is synthesized from a DNA template and forms mRNA
• mRNA carries the genetic code for protein synthesis

Key Enzymes in Transcription

• RNA Polymerase is the main enzyme involved in transcription
• RNA Polymerase synthesizes RNA in the 5' → 3' direction, adding RNA nucleotides complementary to the DNA template strand and recognizes the promoter sequence to start transcription
• Transcription Factors are proteins that help RNA polymerase bind to the promoter region and initiate transcription, also helping regulate gene expression
• Helicase unwinds the DNA at the transcription start site, allowing RNA polymerase to read the template strand
• Topoisomerase relieves supercoiling strain ahead of the RNA polymerase, as it moves along the DNA
• Capping Enzymes add a 5' cap to the mRNA transcript, protecting it from degradation and helping with translation initiation
• Spliceosomes remove introns (non-coding regions) and splice together exons (coding regions) during RNA splicing

Key Steps in Transcription

• Initiation begins when RNA polymerase binds to the gene's promoter region with the help of transcription factors
• DNA unwinds, and RNA polymerase synthesizes the RNA transcript from the 5' → 3' direction
• Elongation proceeds as RNA polymerase adds RNA nucleotides complementary to the DNA template strand where the RNA chain elongates as the polymerase moves
• Termination occurs when RNA polymerase encounters a terminator sequence or a signal, which causes it to release the RNA molecule
• After transcription, the 5' end of the mRNA is capped, and introns are removed by splicing to form mature mRNA
• A poly-A tail is added to the 3' end of the mRNA to protect it and aid in its export from the nucleus by polyadenylation

Translation Overview (RNA to Protein)

• Translation is the process by which an mRNA sequence is used to synthesize a polypeptide (protein) and occurs at the ribosome
• tRNAs bring amino acids to the ribosome in the correct order

Key Elements in Translation

• Ribosomes read mRNA and synthesize the polypeptide chain
• Ribosomes contain two subunits, large and small
• The A site is where the incoming tRNA with an amino acid binds
• The P site is where the growing polypeptide chain is held
• The E site is where the uncharged tRNA exits the ribosome
• Aminoacyl-tRNA Synthetases attach the correct amino acid to its corresponding tRNA molecule, creating a "charged" tRNA
• Initiation Factors are proteins that help the small ribosomal subunit bind to the mRNA and help the large ribosomal subunit assemble for translation
• Elongation Factors are proteins that assist in the polypeptide chain's elongation by facilitating the ribosome's movement along the mRNA
• Release Factors are proteins that recognize the stop codon in the mRNA and cause the ribosome to release the polypeptide chain, terminating translation

Key Steps in Translation

• The small ribosomal subunit binds to the 5' end of the mRNA with the help of initiation factors
• The first tRNA, carrying the amino acid methionine, binds to the start codon (AUG) on the mRNA
• The large ribosomal subunit assembles with the small subunit, and translation begins
• A charged tRNA enters the A site of the ribosome and binds to the codon on the mRNA
• The ribosome moves along the mRNA, transferring the growing polypeptide chain from the tRNA in the P site to the tRNA in the A site
• The ribosome shifts to the next codon, and the process repeats itself
• Termination occurs when a stop codon is reached, release factors bind to the ribosome, and the newly synthesized polypeptide is released

Types of Mutations

• Point Mutations are a change in a single nucleotide
• Silent mutations result in no change in the amino acid sequence
• Missense mutations result in a change in one amino acid
• Nonsense mutations introduce a premature stop codon
• Insertions and Deletions involve the addition or removal of nucleotides, which can lead to frameshift mutations that significantly alter the protein
• The start codon (AUG) begins translation and adds methionine
• Stop codons (UAA, UAG, UGA) end translation
• The codon AUG always codes for Methionine (start codon)
• The codon UUU always codes for Phenylalanine
• The codon UAA is always a stop codon

Bacterial Transcription Factors

• Sigma factor is used in bacterial transcription to help RNA polymerase bind to the -10 and -35 promoter regions
• Eukaryotes use transcription factors and other helper proteins to guide RNA polymerase II to the TATA box instead of sigma factors

Ribosomal Subunits

• A small ribosomal subunit (40S) binds to the 5' cap of the mRNA along with initiation factors (like eIFs) and a special initiator tRNA carrying methionine (AUG)
• The small subunit + tRNA complex scans the mRNA looking for the start codon (AUG), usually the first AUG in a good context (like the Kozak sequence)
• Initiation factors are released when AUG is recognized
• Then, a large ribosomal subunit (60S) joins in, and the full 80S ribosome is assembled
• Translation begins, and the initiator tRNA sits in the P site, ready to go
• mRNA carries the genetic code copied from DNA in the form of codons, which specify the amino acid sequence of the protein
• tRNA matches amino acids to the codons on the mRNA using its anticodon region, bringing the correct amino acid to the ribosome
• rRNA components help form peptide bonds

General Information

• Chromatin contains both protein and DNA
• In translation: Initiation refers to (IFs), Elongation (EFs), and Termination (RFs)
• They are all factors (protein)
• The A site is where the tRNA first binds to the ribosome
• The P site is where the growing polypeptide chain is held
• The E site is where the empty tRNA exits the ribosome after delivering its amino acid
• Bacteria does not have 5’ cap (guanyl transferase), 3’ poly A tail poly (poly A polymerase), or Intron splicing
• Translation: RNA polymerase bumps into a hairpin
• Transcription and translation is coupled in prokaryotes
• New mRNA is transcribed from 5 to 3 direction, as is DNA replication
• Template strand is always 3 prime to 5 prime

Cis- and Trans-Acting Elements

• Cis-acting elements are nucleotide sequences and silencers that lower the rate of transcription of the gene, or to enhancer to activator
• Consensus sequences are shared among bacteria species, with different promoters for bacteria and eukaryotes
• Eukaryotes have many ori while prokaryotes only have oriC
• Cis-acting elements are DNA sequences located on the same DNA molecule (same chromosome) as the gene they regulate and act as binding sites for regulatory proteins, where RNA polymerase binds to start transcription
• Enhancers/Silencers increase or repress transcription of nearby genes, and operator sites in prokaryotes are where repressors bind
• Trans-acting elements are usually proteins that are encoded by genes located on a different chromosome that binds to cis-acting elements to regulate gene expression
• Examples include the Lac repressor protein in E. coli, RNA polymerase, and general or specific transcription factors

Effects on Protein Structure via Mutations

• Silent mutations have no effect on protein structure or function
• Missense mutations may change protein structure, possibly altering function
• Nonsense mutations lead to a truncated protein, which is often nonfunctional
• Frameshift mutations cause extensive changes in the protein sequence, often resulting in a nonfunctional or misfolded protein