Nucleic Acids - Transcription and Translation

Transcription Nucleic acid function: Central Dogma Transcription mRNA structure Stop Codon AUG prokaryotes eukaryotes 5’-UTR Shine-Delgarno OPEN READING FRAME (ORF) …AAAAAAAAAA 3’-UTR 3’-Poly(A) 5’-cap Start of Translation End of Translation 5’ cap and 3’ polyA tail AAGGAGGU poly(A) polymerase (PAP) 5’ poly(A)-binding protein (PABP II) 3’ 2 Transcription Process: •Initiation •Elongation •Termination RNA Polymerases need 3 things: •NTPs •Template •Place to start (NO primer needed) Transcription-Initiation Starts!with!double! stranded!DNA!(gene)! ! Template:!DNA!that! RNA!is!synthesized!to! (Hbonding!partners)! (an5sense,!nonTcoding)! Transcription GCGATATCGCAAA What is the sequence of the newly synthesized RNA strand? 5’-CGCUAUAGCGUUU-3’ ! Coding!(nonTtemplate):! Both strands can be template iden5cal!to!RNA,!with!U! replacing!the!Ts!in!DNA! (sense)! ! Promoters:!sequences! in!DNA!that!aAract!RNA! What about a place to start? polymerase;!similar! Nontemplate strand sequences Always 5’!3’ Promoters Transcription Prokaryotic Transcription – Initiation (a place to start) (Coding strand) Coding strand • RNA pol requires signal to begin transcription • why? • σ factor recognizes promoter region of a gene/operon (–10 & –35 regions) • σ factor-RNA polmerase complex work together to transcribe DNA at specific start sites. • Once σ factor interacts with –10 element, the complex unwinds DNA ~2 turns (open complex). • Called the “Holoenzyme” Coding strand 3 RNAP Complexed With Promoter Coding strand Template strand TranscriptionTranscription Mechanism Inhibitors of Transcription Inhibits elongation at first phosphodiester bond Inhibits elongation by intercalating Inhibits EUKARYOTIC RNAPs (only I & III) Actinomycin D-DNA complex PDBid 1DSC 4 Transcription RNA Polymerase Structure RNA pol is a multi-subunit enzyme (a2bb’ws) These subunits make up the core complex: • two α subunits make non-specific contacts with DNA for positioning • the β and β’ subunits catalyze the addition of ribonucleotides to the growing chain • the ω subunit acts to stabilize the complex Template DNA Nascent RNA Coding strand DNA Subunit Gene MW # Role a2 rpoA 34 2 Non-specific DNA binding b rpoB 150 1 Polymerase b’ rpoC 155 1 Non-specific DNA binding & polymerase w rpoZ 10 1 Zn2+ binding rpoD 70 1 Promoter recognition s RNA•DNA duplex is in the A-form It uses the same mechanism for correct W-C bp and fidelity Rate is ~ 50 base/sec No 3’à5’ exonuclease proofreading Therefore, error rate is the same 1/10,000 Processivity is ~2000 bp Transcription Eukaryotic RNA Polymerase II Conformations Also, the b’ homolog has a disordered CTD that is phosphorylated In going from Initiation to elongation Open conformation Closed conformation Coding strand Template strand 5 11/15/21 Transcription Eukaryotic RNA Polymerase II NTP sites (A & E) i –1 i+1 Bridge A-site Pore E-site Funnel Backtracking RNAP is very sensitive to the helix shape in the active site. If there is a mispaired nucleotide, or even a deoxy nucleotide, it stalls (as well as at damaged DNA). The helix unwinds and the 3’-end of the RNA goes into a P-site. Other subunits/proteins hydrolyze RNA at 3’-end. Proofreading P-site Transcription & Translation Transcription Overview Process RNA Polymerase Fidelity Translation Genetic Code triplet deciphering tRNA Structure Anticodon Acylation (charging) Aminoacyl-tRNA Synthetases Mechanism Fidelity Protein Biosynthesis Overview Process Ribosome review Peptidyl Transferase Fidelity 6 Transcription & Translation Nucleic acid function: Central Dogma Translation: The Genetic Code Recall: Genetic Code is Degenerate & Nonrandom Gold = hydrophobic amino acids; pyrimidine at second position Polar amino acids (blue = basic; red = acidic; purple = uncharged polar) have purine at second position How was the Triplet code discovered? 7 Translation: The Genetic Code ORFs If one base is deleted: If one base is then inserted: THE BIG X RED FOX ATE THE EGG THE IGR EDF OXA TET HEE GG THE IGR EDX FOX ATE THE EGG Brenner & Crick Experiment: The regain of function for the triple mutant told Brenner and Crick that it was a triplet code, uninterrupted. How was the code deciphered? Translation: The Genetic Code Key Developments: 1. Chemical synthesis of nucleic acids 2. in vitro protein synthesis Nirenberg (NIH) 1. First codons used Polynucleotide Phosphorylase (NDP ⇌ RNA + Pi) to make RNA in vitro: poly-A, poly-C, etc. Result: UUU=Phe, AAA=Lys, CCC=Pro, GGG=Gly 2. Chemical synthesis of defined triplets. •Use ribosomes and charged tRNA with different radioactive amino acids •Mix and filtrate - only those amino acids with correct tRNA to complementary “mRNA” will complex with the ribosome Result: 50/64 determined 8 Translation: The Genetic Code Key Developments: 1. Chemical synthesis of nucleic acids 2. in vitro protein synthesis Repeat unit Khorana (MIT) •Chemical synthesis of repetitive RNAs by first making small overlapping complementary DNAs, ligating, and using RNA polymerase to make corresponding repetitive RNAs. •Add synthetic RNAs to in vitro protein synthesis cocktail with radioactive amino acids. •Analyze sequences of the radioactive protein produced. Result: nearly all codons determined, but some remained ambiguous. Combined data from Nirenberg established the CODE. This method was only one able to determine the stop codons. Translation: The Genetic Code Key Developments: 1. Chemical synthesis of nucleic acids 2. in vitro protein synthesis Repeat unit Khorana (MIT) •Chemical synthesis of repetitive RNAs by first making small overlapping complementary DNAs, ligating, and using RNA polymerase to make corresponding repetitive RNAs. •Add synthetic RNAs to in vitro protein synthesis cocktail with radioactive amino acids. •Analyze sequences of the radioactive protein produced. Result: nearly all codons determined, but some remained ambiguous. Combined data from Nirenberg established the CODE. This method was only one able to determine the stop codons. 9