Second Lecture Transcription and Translation (1).pdf

Transcription and Translation Dr N.M.C. Nayanakantha Senior Lecturer (Grade I) Dept. of Biosystems Technology Faculty of Technological  Central Dogma DNA mRNA Protein DNA DNA – Replication – Nucleus DNA mRNA – Transcription – Nucleus mRNA...

Transcription and Translation Dr N.M.C. Nayanakantha Senior Lecturer (Grade I) Dept. of Biosystems Technology Faculty of Technological  Central Dogma DNA mRNA Protein DNA DNA – Replication – Nucleus DNA mRNA – Transcription – Nucleus mRNA Protein – Translation – Ribosome in cytoplasm Central Dogma  Transcription The DNA in genomes does not direct protein synthesis itself, but instead uses RNA as an intermediary. When the cell needs a particular protein, the nucleotide sequence of the appropriate portion of the immensely long DNA molecule in a chromosome is first copied into RNA This is called transcription 4  DNA  mRNA Copying of DNA’s message to mRNA Occurs in the nucleus Pre-mRNA is processed into mRNA and then leaves the nucleus for the cytoplasm (ribosome) Structure of a Gene 6  Transcription in Prokaryotes Prokaryotes: bacteria, blue green algae  Gene transcription is done by a single large enzyme  E. coli RNA polymerase (Holoenzyme)  Transcription takes place in three phases: 1. Initiation, 2. Elongation and 3. Termination. 7 8 1. Transcription Initiation  RNA polymerase enzyme (Holoenzyme) recognizes a specific site on the DNA, called promoter site.  This is found upstream from the gene that will be transcribed,  Then it unwinds the DNA locally. Promoter region Upstream of gene 9 10 The holoenzyme binds to a promoter region about 40–60 bp in size and then initiates transcription a short distance downstream Within the promoter, there are two sequences (6 base pairs length)that are important for promoter function. 1. TATA box (Pribnow box) 2. GACA 11  1. TATA Box (Pribnow Box)  The –10 sequence has the consensus TATAAT sequence  This element was discovered by Pribnow,  Therefore it is also known as the Pribnow box. 2. GACA sequence  The –35 sequence has the consensus TTGACA sequence  This is important in DNA unwinding during transcriptional initiation. 12 13 2. Transcription elongation During elongation the RNA polymerase uses the template strand of DNA and synthesizes a complementary RNA molecule using ribonucleoside 5’ triphosphates (ATP, CTP, GTP, UTP) as precursors. The RNA produced has the same sequence as the non- template strand, called coding strand except that the RNA contains U instead of T. 14 15  3. Transcription Termination  Transcription continues until a termination sequence is reached.  The most common termination signal is a GC-rich region that is a palindrome, followed by an AT-rich sequence.  RNA polymerase “falls off” the DNA strand when the termination sequence (terminator) is reached. 16 17 18  Transcription in Eukaryotes RNA Polymerase II is involved in mRNA synthesis (transcription) 19 In order to initiate transcription, RNA polymerase II requires the assistance of several other proteins or protein complexes, called general (or basal) transcription factors, which must assemble into a complex on the promoter in order for RNA polymerase to bind and start transcription 20  Transcription Transcription Initiation Elongation and Termination  Processing of mRNA  Pre-mRNA mature m-RNA  5’ cap – guanine and phosphate cap on the 5’ end of mRNA  3’ poly-A tail – 200-250 Adenines are added to the 3’ end of the mRNA  Both the 5’ cap and 3’ poly-A tail facilitate the export of mRNA from the nucleus  Both protect the mRNA from degradation by hydrolytic enzymes in the cytoplasm  Both help ribosomes attach to the 5’ ends of the mRNA strand 23 RNA processing  Translation  mRNA  protein  Process of mRNA converting to a protein  Occurs in the cytoplasm – Ribosome 25  tRNA  Translator of mRNA’s message is tRNA – transfer RNA  80 nucleotides long  Hairpin shape – L shaped  One end contains an anticodon which pairs with the codon on the mRNA  Codons determine which amino acid is coded for by the DNA  The other end contains an amino acid attachment site  Aminoacyl-tRNA synthetase attaches the correct amino acid to the tRNA 26 tRNA 27 28  Ribosomes  Pair codons on mRNA with anticodons on tRNA to form polypeptides  Made of large and small subunits  rRNA – ribosomal RNA  Made in the nucleolus  Contain multiple binding sites  mRNA binding site  P site – peptidyl – tRNA site  A site – aminoacyl – tRNA site  E site – exit site 29  1. Translation Initiation Binding of ribosomes onto mRNA strand Shine-Dalgarno 30  Triplet Code: Codons Universal code 3 Nucleotides = 1 amino acid 31 Genetic Code Eg: UUU=Ph e UCC=Ser UAA=Sto p UAG=Sto p UGA=Sto 32 33 2. Translation elongation & Termination 34 Ribosomes 35  Making a protein  Initiation  Small subunit binds to mRNA  Start codon AUG – methionine at P site  Elongation  A site recognizes codon and pairs with correct tRNA  Peptide bond forms between the carboxyl end of the polypeptide at the P site and amino acid at the A site  Amino acid in the A site translocates to the P site  Termination  Stop codon is reached at the A site  UAA, UAG, UGA  Release factors free the polypeptide from the ribosome 36  Making a Protein TRANSCRIPTION DNA mRNA Ribosome TRANSLATION Polypeptide Amino Polypeptide acids tRNA with amino acid Ribosome attached Gly tRNA Anticodon A A A U G G U U U G G C 5 Codons 3 mRNA  Proteins  Fold spontaneously into primary, secondary, and tertiary structures.  Chaperone proteins assist in folding. 38