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CHAPTER 4, PART 2 FLOW OF GENETIC INFORMATION CHAPTER 4, PART 2: FLOW OF GENETIC INFORMATION LECTURE TOPICS 1. RNA properties in general 2. Transcription: RNA Polymerase 3. mRNA properties 4. Genetic Code 5. Eucaryotic genes: structural organization RNA is usually a single-stranded polynucleotide Single-Stranded DNA and RNA can form helical hairpins Complementary when reversed (fold back and base pair) 3` 5` 3` 5` 5` 3` 5` 3` Stem-loop (hairpin) structures [Fig.5.19] Typical highly base-paired RNA structure * 3 bases form H-bonds ends * [p.126 and Fig.5.20] * * * * * (----) Extra H-bonds (----) Watson-Crick H-bonds in (G-C) base pair Types of cellular RNA molecules • All RNA in E. coli (a bacterium) is made by one RNA polymerase • mRNA encodes protein amino acid sequences The “Central Dogma” of molecular biology 10 -4,-5 10-3, -4 10-8 Transcription translation DNA RNA Replication Reverse transcription DNA virus Retrovirus PROTEIN RNA Virus Prions 10-4 FEATURES OF PROCESSES ¾ Accuracy - RELATIVE = error rates ¾ Signals - STARTS AND STOPS ¾ Stages - INITIATION, ELONGATION, TERMINATION 2) TRANSCRIPTION: DNA Æ RNA RNA polymerase makes RNA that is complementary to a DNA template strand Coding strand DNA sequence is same as mRNA (or any RNA transcript), but has T’s instead of U’s TRANSCRIPTION: DNA Æ RNA RNA polymerase: DNA-dependent enzymes which require: A. A template DNA (preferably double-stranded) B. Activated precursors (all 4 rNTPs – ATP, GTP, CTP, UTP) C. Mg++ D. Reaction is: (RNA)n + rNTP (RNA)n+1 + PPi 2Pi E. Same reaction mechanism as DNA polymerase F. RNA polymerase does not require a primer RNA polymerase reaction mechanism: (same as DNA polymerase) 3`-OH attacks α-P 3` 5` Nucleophilic attack 3` 5` New 3`Æ5` phosphodiester bond • Sequences shown are DNA Coding Strand • Sequences shown are same as RNA but T’s instead of U’s TRANSCRIPTION START SIGNALS: Promoters TRANSCRIPTION TERMINATOR: Stop Signal “Signal” is in RNA transcript base sequence and structure G-C rich “Stem” (D.S.) 5` 3` Pyrimidines – usually U 3) mRNA (messenger RNA) Jacob and Monod predicted properties of mRNA: 1. A polynucleotide (RNA) 2. Base composition complementary to a DNA template 3. Size variation reflects variety of protein sizes (3 bases/a.a.) 4. Transient association with ribosomes 5. Rapid turnover (~2 minute half life in E. coli) T2 Bacteriophage used to learn mRNA properties E. Coli cell After infection, Cell makes: 1. DNA 2. RNA [FAST, after 1] 3. viral proteins 4. new virus mRNA: Experiment to verify predictions Known: T2 bacteriophage infection is followed by synthesis of RNA, proteins, and new virus particles. Experiment: • • Infect E. coli with T2 phage (add 32P-phosphate at same time) Study properties of polynucleotides that incorporate 32P-phosphate. Proof that new RNA is complementary to T2 DNA D.S. HYBRID 3H 3H 3H 1) HEAT 3H 32P 3H 3) COOL + 2) Add T2 DNA 32P Result shows that T2 DNA has genes that code for the new RNA New RNA mRNA ? What if you use E. coli DNA instead of T2 DNA? T2 DNA T2 Bacteriophage result: mRNA: Base composition is complementary to DNA template E. Coli cell After infection, Cell makes: 1. DNA 2. RNA [FAST, after 1] 3. viral proteins 4. new virus CHAPTER 4, PART 2: FLOW OF GENETIC INFORMATION LECTURE TOPICS 1. RNA properties in general 2. Transcription: RNA Polymerase 3. mRNA properties 4. Genetic Code 5. Eucaryotic genes: structural organization The “Central Dogma” of molecular biology 10 -4,-5 10-3, -4 10-8 Transcription translation DNA RNA Replication Reverse transcription DNA virus Retrovirus PROTEIN RNA Virus Prions 10-4 FEATURES OF PROCESSES ¾ Accuracy - RELATIVE = error rates ¾ Signals - STARTS AND STOPS ¾ Stages - INITIATION, ELONGATION, TERMINATION Translation (RNA Æ Protein) tRNA - adaptors that deliver a.a`s to the translation system “transfer” RNA Base to a.a. code * Codon (mRNA) 3` 2` * Anticodon (tRNA) Amino Acid Aminoacyl-tRNA Base pairs with mRNA codons THE GENETIC CODE (1964) 1) 3 letters 2) Sequential 3) Non-overlapping 4) No “punctuation” “Colinear” Q? What if code read /BCD/EFG/….etc THE GENETIC CODE The Genetic Code: How many code words (codons)? [43 = 64 codons of 3 bases each (all are used)] [“The Universal Code”] Arg = CG(N) (Start) AUG TRANSLATION: Start signals on mRNA Mitochondrial Genetic Code has differences [“Universal”] * * GENETIC CODE: Summary A. A codon (3 bases) specifies an amino acid B. Sequential and non-overlapping C. Degenerate (more than one codon/amino acid) D. Some codons are start and stop signals E. The code is nearly universal (see differences in human mitochondrial code) F. Sequences of bases in genes and amino acids in their encoded proteins are colinear G. Experiments with synthetic mRNAs established codon assignments CHAPTER 4, PART 2: FLOW OF GENETIC INFORMATION LECTURE TOPICS 1. RNA properties in general 2. Transcription: RNA Polymerase 3. mRNA properties 4. Genetic Code 5. Eucaryotic genes: structural organization Prokaryotic mRNA base paired to its gene DNA-RNA hybrid, base-paired A continuous coding sequence on DNA Eukaryotic mRNA base paired to its gene β- globin mRNA-DNA hybrid D.S. DNA DNA-RNA hybrid loops Eucaryotic mRNA base-paired to its gene (D.S. DNA) (RNA-DNA D.S. hybrid) (D.S. DNA) DNA coding sequence must be discontinuous! Eukaryotic gene transcription and mRNA maturation GENE X1 X2 X3 X = exon Pre-mRNA X1 X2 X3 Removal of introns + mRNA X1 X2 X3 All 3 exons joined RNA intron splicing signals: • Conserved in all eukaryotes. • Occur at or near intron-exon junctions A 5` 3` Modified ends of Eukaryotic mRNAs [Post-transcriptional] N7-MeG Protein A mature mRNA Why introns? One possibility is to facilitate evolution of new proteins by exon shuffling New genes code for proteins with a mix of exons from pre-existing genes to get proteins with new functions. Alternative Splicing can result in mRNA’s for different proteins from same primary transcript (Ex: membrane-bound vs soluble antibody) 1 2 3 Exon Intron 1 2 3 4 4 5 A Alternative Splicing 5 Spliced mRNAs 6 6 Exons 6 Pre- mRNA for antibody gene B 2 3 4 5 Exons 5 6 PROKARYOTE DNA EUKARYOTE DNA Exon Intron Exon Pre m-RNA Splice out intron m-RNA m-RNA Protein Protein Eucaryotic Genes: Summary of Structural organization 1. Allmost all have coding sequences (exons) interrupted by noncoding sequences (introns) 2. After transcription, introns are removed and exons are joined accurately by splicing at evolutionarily conserved sequences. 3. Exon polarity (5`Æ3`) is retained after splicing 4. Protein domains coded by exons can be rearranged to give proteins with new functions 5. Alternate splicing of an mRNA can give different proteins. (Ex: membrane-bound vs soluble antibody) 6. Almost all bacterial genes lack introns (had but lost during evolution to adapt to very rapid growth) CHAPTER 4, Part 2 Study Exercise 1. Draw representative prokaryotic and eukaryotic genes 2. Indicate locations of signals for transcription and translation, relative to each other 3. For a eukaryotic gene, indicate intron processing signals and their location, relative to other signals