Module 3 _ Central Dogma (1).pdf

Module III: The Central Dogma Lecture Outline 1. DNA Replication 2. RNA Transcription 3. Protein Translation How much do you remember about the central dogma? DNA is the Heritable Material 1944: O. Avery, C. MacLeod, M. McCarty 1. 2. 3. Worked with a batch of heat-killed S strain (pathogeni...

Module III: The Central Dogma Lecture Outline 1. DNA Replication 2. RNA Transcription 3. Protein Translation How much do you remember about the central dogma? DNA is the Heritable Material 1944: O. Avery, C. MacLeod, M. McCarty 1. 2. 3. Worked with a batch of heat-killed S strain (pathogenic) bacteria. They divided it into 5 batches. In the first batch, they destroyed the polysaccharide coat of the bacteria; in the second batch they destroyed its lipid content; they destroyed the RNA of the bacteria in the third batch; with the fourth batch, they destroyed the proteins; and in the last batch, they destroyed the DNA. Each of these batches was individually mixed with live R (non-pathogenic) strain bacteria and injected into individual mice table al herter d From all 5 mice, all of them died except the last mouse. From all the dead mice, live S strain bacteria was retrieved. ~ 0 How does ANS:It Two possible heritable? DNA become replicates. events: parent ↳Conservative: & I semiconservative:I parent 4 daughter DNA STRAND & -E DNA Replication I daughter 3 strand DNA Replication DNA Replication is Semiconservative -> Parent E daughter RESULT: 1 DNA Replication requires DNA Polymerase • DNA polymerase is the enzyme responsible for template synthesizing DNA using a ↳DNA template phosphate • 2 Fundamental Properties: group 1. Synthesize DNA in the 5’ -> 3’ direction 2. Can only add new deoxyribonucleotides to preformed primer that is H-bonded to the template strand Primase: Enzyme responsible for synthesizing short RNA fragments to act as primers. ther bonds:dies DNAnO proods 1 Association of DNA polymerase onto the DNA How the the does the DNA efficient DNA polymerase and remain more through associated itself with urroundtre DNA? Sliding Clamp - Proliferating Cell Nuclear Antigen [PCNA] • Increase activity of polymerases and causing them to remain bound to DNA • PCNA – load the polymerase onto the primase and maintain association with the template survei ↓ DNA Spuroug d strar openthe Proper once released X • Replication Factor C [RFC] – Clamp-loading proteins that loads PCNA onto the DNA (ATP hydrolysis) the I recruits Polymerase, DNA allowing to be close and make more it it efficient 1 DNA Replication Requires Other Proteins sequence & Onsite Origin of Replication (Ori) - opening verSara binds andfurther opens up DNA (SSD) stance singue ↳ binds prevents -> venlybridnation Primace:Adding DNA purers on strand parental RNA to Role of Topoisomerase Helicase: Catalyzes the unwinding of parental DNA, coupled to the hydrolysis of ATP at the replication fork Single-stranded DNA-binding proteins: stabilize the unwound template DNA, keeping it in an extended single-stranded state so that it can be copied by the DNA polymerase topoisomerase ↳relives Topoisomerase: Enzymes catalyzing reversible breakage and joining of DNA - Topoisomerase I and II tension tension 1 Active Sites of DNA Replication Replication Fork Replication ↳ 2 ends fork where helicase • Regions of Active DNA synthesis I fo rks • 2 Replication forks in 1 Replication Bubble DNA can replication have multiple bubbles 1 They DNA the along are recognized by helicase allowing strands Parent single-stranded parent strand to begin DNA. and further to reparate, RNA pumers SSD bind on the of RFC allows the primed onto the dessociates. PCNA recruits DNA sliding DNA. polymerase. Bringing it all together: DNA the polymerase has DNA polymerase producing bubble from is sto 3 continues bonds attaching by a free 3' OH. bound, it direction. Once can start Replication grow. to DNA opens the DNA. RNA primers. OH endsbecause is primed, DNA associate to clamp RFC a primes Once profens, initiator bind primers. Free ORIs. are RNA on Replication The Strands of DNA Replication The Leading and Lagging Strand Leading Strand: Strand that is synthesized continuously, in the direction of the replication fork. Lagging Strand: Strand that is synthesized in small pieces, “opposite” direction as the replication fork Okazaki Fragments: Small Segments of newly synthesized DNA. 1 Different Families of DNA Polymerase Primase: Enzyme responsible for synthesizing short RNA fragments to act as primers. • In Bacteria: • DNA Polymerase III is the primary DNA used for replication DNA Polymerases ~Transigen s • Eukaryotic cells: • DNA polymerases (α, δ, and ε – and β) that function in replication of nuclear DNA. gamma • DNA polymerase γ is localized to mitochondria and is responsible for replication of mitochondrial DNA 1 DNA Replication Requires Other Proteins RNA polymerases recognizes nucleotide pairings. Changes MDNA by RNA shape is recognized polymerase as a proofread Removal of RNA nucleotides: - In bacteria: DNA Pol I acts as a polymerase and as an exonuclease X recognizes - In Eukaryotes: RNase H + 5’ -> 3’ exonuclease, which degrades the RNA in RNA-DNA hybrids - DNA Ligase: Enzyme facilitating DNA (or Okazaki) fragment joining 2 The Fidelity of DNA Replication 1 DNA Replication Maintaining the end of the chromosomes - Sequences added of DNA the end, at Telomeres: tandem repeats of simple-sequence DNA Telomerase: catalyzes the addition of telomeres, able to catalyze synthesis of telomeres in the absence of a template - type of reverse transcriptase that carries its own RNA template everytime It can replicates, DNA pol shortens, because synthesize only from 5-3' TTAGL Telomeres: lifespan of the DNA 1 B. Transcription Overview: The Flow of Genetic Information • Gene expression, the process by which DNA directs protein synthesis, includes two stages: transcription and translation • Transcription is the synthesis of RNA under the direction of DNA • Translation is the synthesis of a polypeptide, using information in the mRNA © 2012 Pearson Education, Inc. 0 DNA template strand Transcription 3¢ 5¢ A C C A A A C T T G G T T C G A G G G C T T C A 5¢ 3¢ DNA molecule Gene 1 TRANSCRIPTION Gene 2 mRNA U G G 5¢ U U U G G C U C A 3¢ Codon TRANSLATION Protein Trp Amino acid Phe Gly Ser Gene 3 0 Transcription requires RNA Polymerase • Transcription is the DNA-directed synthesis of RNA • RNA nucleotides are linked by the transcription enzyme RNA polymerase. ↳ Reads template At a & DNA as a creates RNA 1 to 3' E coli RNA Polymerase: ↳ 6 subunits:'M, O, a da, z Families of RNA Polymerases in eukaryotes: RNA Pol II – transcribes protein coding genes, along with miRNAs and lncRNAs RNA Pol I and III – Transcribes Ribosomal RNAs (rRNAs) and transfer RNAs (tRNAs) © 2012 Pearson Education, Inc. 2 Transcription is the DNA-directed Synthesis of RNA The DNA sequence where RNA polymerase attaches is called the promoter; the sequence signaling the end of transcription is called the terminator The stretch of DNA that is transcribed is called transcription unit ↳ in between promoter d Steps: terminator 1. Transcription begins with initiation, as the RNA polymerase attaches to the promoter. 2. During the second phase, elongation, the RNA grows longer. 3. Finally, in the third phase, termination, the RNA polymerase reaches a sequence which signals the end of transcription. © 2012 Pearson Education, Inc. 2 Transcription – Initiation + Elongation (Prokaryotes) Recognized RNA by the polymerase Initiation occurs in the promoter region: 3 S • From -35 to -10 ↳A • +1 Transcription start site 35 - - - 100 - 7O 10 begins start + 10 site gi 100 + polymerase transcription Steps: 1. Sigma recognizes -35 and -10 and forms the closed- RNA polymerase binds to the DNA promoter complex 2. DNA is unwounded (open promoter complex) 3. Initiation of Transcription 4. After ~10 nts transcribed, sigma is released ↳happens eukaryotes 5. B + B’ grips the DNA In generals 6. Elongation to Side Note: Initiation of Transcription in Eukaryotes require Transcription Factors (see Module IV) 2 Transcription – 2 Types of Termination How does the RNA polymerase know when to terminate? A. Rho-Independent Termination consensus sequence >"Termination sequence" strong ) estable hydrogen bond - Inverted repeats create a symmetrical GC Hairpin-loop Formation - Followed by 7 U’s 2 Transcription – 2 types of Termination Ruo protein B. Rho- Dependent Steps: 1. Rho Sequence binds to the mRNA 2. Moves along mRNA 5’ -> 3’ 3. Rho acts as a helicase and separates RNA from DNA 2 Transcription of Pre -mRNA to mRNA Pre-mRNA to mRNA 1. Addition of 5’ Cap 2. Splicing of introns (Module IV) 3. Poly-A tail Three typical processing steps Transcription of Pre -mRNA to mRNA X T il I ,a red TriphOSP a Pre-mRNA to mRNA 5’ G-Cap Fa cilitated by: capping enzyme Methyltransferase Pre-mRNA to mRNA - splicing site ↓splicing Splicing ↳ Somewhere the intron Adenine attached RNA Components (small nuclear RNA – snRNA): U1, U2, U4, and U5 -> complexed with 6-10 proteins forming small nuclear ribonucleoproteins - snRNP Different types: UI, an its branching point Spliceosome – RNA and protein complex that facilitates pre-mRNA processing. -> as along to U2, 44/Ub, Us site Pre-mRNA to mRNA Splicing (in more Details) recognizes binds a beingsite to replaced by no - carrier ↳ protein and for 46 dissociates along recognizes -> the ligates branch point - - 44 with i to the branch point this storature catalyzes, S dimer Introns larat ↳ dissociates with sURPs E binds to the secondary structure along ↓ keeps ends closed together 1 X recognizes CPSF 00 ↳ cleavage factor cleares C ⑳ ↳ template PABP Pre-mRNA to mRNA Polyadenylation of the 3’ end Facilitated by: • CPSF - cleavage and polyadenylation factor • cleavage factor • PolyA polymerase protein independent Protein Translation Protein translation DNA template strand 3¢ 5¢ A C C A A A C T T G G T T C G A G G G C T T C A 5¢ 3¢ DNA molecule Gene 1 TRANSCRIPTION Gene 2 mRNA U G G 5¢ U U Protein 3¢ that Codon TRANSLATION U G G C U C A 3 nucleotide bases base, code for specific Triplet Trp Amino acid Phe code Gly Ser Gene 3 0 Amino Acids 0 Translation Overview • Translation is the RNA-directed synthesis of a polypeptide • The language of mRNA molecule is translated into the language of polypeptide. • Characteristics of the genetic code • Three nucleotides specify one amino acid. 64 • • • 61 codons correspond to amino acids. AUG codes for methionine and signals the start of transcription. 3 “stop” codons signal the end of translation. © 2012 Pearson Education, Inc. 3 Genetic code Dictates Translation of Amino Acids The genetic code is • redundant, with more than one codon for some amino acids, • unambiguous in that any codon for one amino acid does not code for any other amino acid, • nearly universal—the genetic code is shared by organisms from the simplest bacteria to the most complex plants and animals, and • without punctuation in that codons are adjacent to each other with no gaps in between. © 2012 Pearson Education, Inc. 3 Molecular Components Translation Molecular Components of of Translation § Messenger RNA (mRNA) • encodes amino acid sequences and • The genetic message is read in consecutive groups of three letters based on the genetic code © 2012 Pearson Education, Inc. Poly Binding A Proten (PABP) 3 Molecular Components Translation Molecular Components of of Translation for hydrates re § Transfer RNA (tRNA) molecules function as a language interpreter, >Anticodon ↳ read 3) to I • converting the genetic message of mRNA • into the language of proteins. § Transfer RNA molecules perform this interpreter task by • picking up the appropriate amino acid and • using a special triplet of bases, called an anticodon, to recognize the appropriate codons in the mRNA. © 2012 Pearson Education, Inc. 3 tRNA and Animo-acyl Synthetases § Amino-acyl Synthetases: group of enzymes that facilitates transfer of amino acids to tRNAs § 2 step process: 1. Amino acid is activated 2. 3’ Terminus joining © 2012 Pearson Education, Inc. 3 Molecular Components of Translation Molecular Components of Translation Ribosome synthesizes polypeptide chain. • The two ribosomal subunits (large and small) are made of proteins and ribosomal RNA (rRNA) • Ribosomal subunits come together during translation. • Ribosomes have binding sites for mRNA and tRNAs. © 2012 Pearson Education, Inc. 3 Building an Amino Acid Polypeptide Chain Building a polypeptide chain § Translation can be divided into the same three phases as transcription: 1. initiation, · · · 2. elongation, and · 3. termination. © 2012 Pearson Education, Inc. 3 Initiation of Translation Terms to know: XXAVE & - 5ʹ untranslated regions (UTR) - Messenger RNAs that encode multiple polypeptides are called polycistronic and - monocistronic mRNAs encode a single polypeptide chain prokaryotes - Shrine-Dalgarno Sequence 3 Initiation of Translation - Components Eukaryotic Initiation Factors (eIFs) I only present in eukaryotes With meth-tRNA: eIF2 (with GTP) With mRNA: - eIF4E (recognizes the 5’ Cap) - eIF4A and 4G (recognizes the IRES) - 4G also binds PABP - PABP – binds 4G and Poly A tail eIF5: complexes with 40S and meth-TRNA, facilitates hydrolysis of eIF2GTP eIF5B – Binds 60s with 40S Side Note: Internal Ribosome Entry Sites (IRES) 3 e / associated I 400s with eIF Xsubunit crea teaes mRNA more & efficient because es of 40stRNmex Met-TRNA LOOP disSO cat hydrolysis associated with 40S leaving babiesreconcie behind this as es & W ass,see I instate recognizes asso cates Itself with each other s binds with -> cap in Initiation of Translation (Bringing it all Together) Eukaryotic initiation factor 3 Elongation of Translation 40s & 60s subunit complex 3 ribosomal sites: E - exit P - Peptidyl A – aminoacyl Steps: 1. meth-tRNA bound at P site 2. Aminoacyl-tRNA enters A site (escorted by eEF1a-GTP) P E A 3. movement of forward 4. 5. If anti-codon and codon matches, eEF-GTP hydrolysis Transfer of methionine (or growing polypeptide chain) to aminoacyl tRNA at the A site Translocation (Hydrolysis of eE2) 6. 7. tRNA exits Repeat 2-7 facilitates ribosomes ↳ 3 Termination of Translation - Elongation continues until the presence of a stop codon - Release Factors recognize Stop codons - Hydrolysis of the bond between polypeptide and tRNA 3 Recap of Gene Expression Transcription + Translation Implications DNA Replication – Fidelity (SNPs) Viruses – How they hijack the central dogma, How they are detected, + Antivirals (Eliott Gertrude) Exceptions to the rule - Reverse Transcriptase (RNA to DNA) - Prions (protein to protein) 4