--- EUKARYOTIC TRANSCRIPTION Process: DNA → pre-mRNA → (processing) → mature mRNALocation: Nucleus --- 1. Initiation Phase - Transcription starts when the enzym
--- EUKARYOTIC TRANSCRIPTION Process: DNA → pre-mRNA → (processing) → mature mRNALocation: Nucleus --- 1. Initiation Phase - Transcription starts when the enzyme RNA polymerase II needs to attach to the DNA at the start of a gene . - It looks for a special DNA sequence called the promoter . - Inside this promoter, there's a key signal called the TATA box , usually found about 25 bases before the actual starting point of transcription. - But RNA polymerase II can’t bind to the DNA by itself — it needs help . - First, a helper protein called TFIID comes in. Inside TFIID is a special subunit called TBP ( TATA-binding protein ) — this is the one that sticks to the TATA box . - After TFIID binds, more helper proteins join in — these are other transcription factors like TFIIA and TFIIB . - Together, all these proteins and RNA polymerase II form a group called the pre-initiation complex (PIC) . - This complex makes sure that transcription starts at the correct point on the DNA. --- 2. Elongation Phase - Once the complex is ready, the DNA opens up at the transcription start site — this creates a bubble where transcription happens. - RNA polymerase II begins reading the template DNA strand from 3' to 5' direction . - It builds the new RNA strand in the 5' to 3' direction . - It adds free RNA building blocks called nucleoside triphosphates (NTPs) — these are: ATP , UTP , GTP , and CTP . - These are added one by one , matching the DNA bases (A with U, T with A, G with C, C with G). - As RNA polymerase II moves along the DNA:It unwinds the DNA ahead . - It closes it back behind , keeping the bubble moving forward. --- 3. Termination and Processing - Transcription ends when RNA polymerase II reaches a termination signal — this is a special sequence in the RNA: AAUAAA (called the polyadenylation signal). - At this point, the pre-mRNA is released — but it's not ready to be used yet. - It needs to go through processing steps to become mature mRNA : 5' Cap Addition :A 7-methylguanosine cap is added to the 5' end . - This protects the RNA and helps it bind to ribosomes later. - 3' Poly-A Tail Addition :A long chain of adenine nucleotides (poly-A tail) is added to the 3' end . - This gives stability and helps in translation efficiency . - Splicing : Introns (non-coding parts) are cut out . - Exons (coding parts) are joined together . - The final mature mRNA now leaves the nucleus and goes to the cytoplasm for translation. --- EUKARYOTIC TRANSLATION Process: mRNA → proteinLocation: Cytoplasm (on ribosomes) --- 1. Initiation Phase (Simplified) - Translation starts when the small ribosomal subunit (40S) binds to the 5' cap of the mature mRNA. - This step needs help from initiation factors called eIFs . - The 40S subunit moves along the mRNA from 5' to 3' direction , looking for the start codon (AUG) . - AUG codes for the amino acid methionine — this marks the true starting point. - A special initiator tRNA , carrying methionine, binds to the AUG codon by base-pairing its anticodon to the mRNA codon . - Once everything is aligned, the large ribosomal subunit (60S) joins. - Together, they form the full 80S ribosome , which has 3 sites : A site – where new tRNA with amino acid enters. - P site – where the growing protein chain is held. - E site – where empty tRNA exits the ribosome. --- 2. Elongation Phase - The ribosome reads the mRNA codon by codon . - For each codon:A matching aminoacyl-tRNA enters the A site . - A peptide bond forms between the new amino acid (in A site) and the existing chain (in P site).This is done by peptidyl transferase , an enzyme built into the ribosome. - The ribosome shifts forward by one codon ( translocation ).The tRNA in P site moves to E site and exits. - The tRNA in A site moves to P site , carrying the growing protein. - This cycle repeats, adding one amino acid at a time to the chain. --- 3. Termination and Post-translational Processing - Translation ends when the ribosome reaches a stop codon — UAA, UAG, or UGA . - These codons don’t code for any amino acid. - Instead, special proteins called release factors (eRFs) bind to the A site . - These trigger the release of the finished polypeptide chain from the ribosome. - The ribosome then breaks apart , ready for another round of translation. - The new protein may still need processing before it's fully functional: Folding (with help from chaperone proteins ). - Cleavage of signal sequences. - Chemical modifications : Phosphorylation - Glycosylation - Acetylation - These steps help the protein get its final shape , proper location in the cell, and biological activity . --- Quick Memory Aid: --- DNA REPLICATION – STEP BY STEP DNA replication is the process by which a cell makes an exact copy of its DNA before division. It is semi-conservative , meaning each new DNA molecule has one old (parental) strand and one new (daughter) strand . --- GENERAL DNA REPLICATION STEPS (Eukaryotes) --- Step 1: Initiation – DNA Unwinding What happens: - The enzyme DNA helicase attaches to the origin of replication (a special DNA sequence). - Helicase breaks hydrogen bonds between base pairs ( A-T and G-C ) and unzips the DNA . - This creates a Y-shaped area called the replication fork . Key points: - Origins are A-T rich (A-T has only 2 hydrogen bonds = easier to break). - Both strands separate and now act as templates . - Replication happens in both directions → bidirectional replication . --- Step 2: Stabilization of Single Strands What happens: - Single-strand binding proteins (SSBs) attach to the separated strands to keep them apart . - DNA gyrase (a type of topoisomerase) removes twists and relieves tension ahead of the fork. Key points: - SSBs prevent strands from reannealing (coming back together). - Gyrase stops supercoiling , making it easier for helicase to move. - Keeps DNA open and stable for replication. --- Step 3: Primer Synthesis What happens: - RNA primase lays down short RNA primers (about 8–12 nucleotides). - These primers provide a 3’-OH end that DNA polymerase needs to begin synthesis. Key points: - Primers are made of RNA , not DNA. - Leading strand → only one primer needed. - Lagging strand → needs many primers . - All new DNA is built in the 5’ to 3’ direction . --- Step 4: DNA Synthesis – Leading Strand What happens: - DNA polymerase starts adding DNA nucleotides to the 3’ end of the primer. - Synthesis goes continuously in the 5’ to 3’ direction , same direction as the fork. - DNA is copied smoothly into one long strand . Key points: - Fast and efficient. - No breaks or fragments — just one continuous piece . - Reads template strand 3’ to 5’ . --- Step 5: DNA Synthesis – Lagging Strand What happens: - DNA polymerase builds DNA in short segments called Okazaki fragments (1000–2000 bp long). - These are made away from the replication fork (still 5’ to 3’). - Multiple primers are needed — one for each fragment. Key points: - Discontinuous synthesis. - Each fragment must be processed and joined later. - More complex than leading strand. --- Step 6: Primer Removal and Gap Filling What happens: - DNA polymerase I removes the RNA primers . - At the same time, it fills in the gaps with new DNA nucleotides . Key points: - RNA is completely replaced with DNA. - Ensures the DNA is pure and ready for sealing. - Leaves small “nicks” (unsealed bonds) between fragments. --- Step 7: Ligation What happens: - DNA ligase seals the final gaps by forming phosphodiester bonds between fragments. - This joins all Okazaki fragments into a single, continuous strand. Key points: - Final sealing step. - Result = two identical DNA molecules . - Each new DNA has one original strand + one new strand → semi-conservative replication . --- POLYMERASE CHAIN REACTION (PCR) – STEP BY STEP PCR is a laboratory technique used to amplify specific DNA sequences exponentially in vitro.Invented by Kary Mullis in 1983.Requires DNA template , primers , Taq DNA polymerase , dNTPs , and a thermocycler . --- PCR Steps (3 Main Phase