Chapters 5, 28, and 29
Chapters 5, 28, and 29 BCM 475 - M001
Popular in Biochemistry I
Popular in Biochemistry
BCM 475 - M001
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Chapter 5 [Pg. 139169] The Exploration of genes Relies on Key Tools 1) RestrictionEnzyme Analysis 2) Blotting Techniques: southern, Northern, Western Blot 3) DNA Sequencing 4) SolidPhase Synthesis of Nucleic Acids 5) PCR: Amplification of a Segment of DNA Restriction Enzymes Split DNA into specific fragments Restriction enzyme = restriction endonucleases Recognize and cleave specific places in double helical DNA Almost always posses twofold rotational symmetry Recognized sequence is Palindromic (inverted repeats) Fragments can serve as fingerprint of a DNA molecule Restriction Fragments can be Separated by Gel Electrophoresis and Visualized phosphodiester backbone is negatively charged, making gel electrophoresis a suitable technique Polyacrylamide gel separate fragments by size ~1000 bp Agarose gel more porous, for larger fragments as large as ~20,000 bp Ethidium bromide or Sybr Green fluorescent dye Blotting Techniques Southern blotting DNA – Hybridize short DNA from a gene to find larger gene fragments Transferrring DNA by blotting a nitrocellulose sheet and add 32Plabeled DNA probe Autoradiography Reveals the position of the restriction [Gragemntprobe duplex] Northern blotting RNA Western blotting Protein DNA can be Sequenced by Controlled Termination of Replication Sanger dideoxy method: Using DNA Polymerase I Labeled dATP, TTP, dCTP, dGTP Dideoxy analog of dATP The strand cannot be extended past the dideoxy analog Each dideoxy nucleotide has a different colored fluorescent dye attached to the base Resulting fragments are separated by Capillary Electrophoresis As DNA fragment emerge form capillary, base sequence can be determined by the fluorescence reads length of 100600 nt DNA Probes and Genes can be Synthesized by Automated SolidPhase Methods Base protected deoxyribonucleoside 3’ phosphoramidites SolidPhase Oligonucleotide Synthesis The activated monomer added to the growing chain is a deoxyribonucleoside 3’phosphoramidite containing a DMT protecting group on its 5’oxygen atom, a BCE protecting group on its 3’ phosphorous oxygen atom, and a protecting group on its base This method can synthesize ~100 nucleotides DNA chains Selected DNA sequences can be greatly amplified by the polymerase chain reaction PCR to amplify specific DNA sequence by adding: A pair of primers that hybridize with the flanking sequencesA of the target All four deoxyribonucleoside triphosphates (dNTPs) and, A heatstable DNA Polymerase 3steps: 1) Strand separation two strands of the parent DNA molecule are separated by heating the solution 95 degree C for 15 sec 2) Hybridization of Primers solution is cooled to 54 degree C to allow each primer to hybridize to a DNA strand 3) DNA synthesis the solution is heated to 72 degree C. Enzyme Taq DNA Polymerase is added to elongate both primers in the direction of the target sequence PCR is a Powerful Technique in Medical Diagnostics Forensic, and Studies of Molecular Evolution The tools for recombinant DNA technology have been used to identify disease – causing Mutations Polymorphisms (genetic variations) may themselves cause disease or be closely linked to another genetic alteration that does Ex: (RFLPs) Restrictionfragment length polymorphisms Recombinant DNA Technology Has Revolutionized All Aspects of Biology Restriction enzymes and DNA ligase are key tools in forming recombinant DNA molecules Vector a DNA molecule that can replicate autonomously in an appropriate host (Choices: plasmid or Lambda Phage) Complementary SingleStranded Ends the stagger cuts made by restriction enzyme “cohesive” or “sticky” ends Can anneal DNA fragments and rejoin with DNA ligase Energy source ATP or NAD+ is required DNAlinker can be cleaved by restriction enzyme Cohesive ends can be formed by the addition and cleavage of a chemically synthesized linker Plasmids and Lamdaphage are choice vectors for DNA Cloning in Bacteria Useful Plasmid Cloning Vectors: An origin of replication A selectable marker (antibiotic resistance gene) A cloning site Polylinker contains 2+ different sites for cleavage by restriction enzymes, making cloning easier by providing sites for DNA fragments to be inserted Lambda Phage destroy its host or can become part of the host Lytic pathwayviral functions are fully expressed: viral DNA and proteins are quickly produced and packaged into virus particles lysis and appearance of virions Lysogenic pathway the phage DNA becomes inserted into the host cell genome and can be replicated together with hostcell DNA for generations Mutant Lambda Phage can be constructed, easier to enter bacteria than do plasmids Cosmid A hybrid of lambda phage and a plasmid that can serve as a vector for large DNA inserts Bacteria and Yeast Artificial Chromosomes (BACs) or (YACs) YAC Contains a centromere, an autonomously replicating sequence (ARS) where replication begins, a pair of telomere, marker genes, and cloning sites Specific Genes can be Cloned from Digests of Genomic DNA To clone gene that’s present just once in a genome: 1) Need a specific oligonucleotide probe 2) A DNA library that can be screened rapidly Creation of genomic library Digest genome The DNA fragment is inserted into the lambda phage vector. Packaging into virions and amplification by infection in E.Coli Rapid genomic screening can be accomplished by DNA hybridization Complementary DNA Prepared from mRNA can be Expressed in Host Cells Bacteria don’t have splicing machinery Enzyme Reverse Transcriptase Synthesizes a DNA strand complementary to an RNA template if the transcriptase is provided with a DNA primer that is base paired to the RNA and contains a free 3’OH group Use Oligo (T) primer DNA is resistant to alkaline hydrolysis, can digest mRNA Enzyme terminal transferase adds nucleotides Formation of a cDNA duplex a complementary DNA (cDNA) duplex is created from mRNA by using reverse transcriptase to synthesize a cDNA strand, first along the mRNA template and then, after digestion of the mRNA, along that same newly synthesized cDNA strand Expression Cloning a radioactive antibody specific for the protein of interst can be used to identify colonies of bacteria that express the corresponding protein product (Can be used when Protein and antibody are expressed and availbale) Proteins with New Functions can be Created through Direct Changes in DNA (In Vitro) Deletion Removing of DNA Substitutions OligonucleotideDirected Mutagenesis: 1) Have plasmid containing the gene or cDNA for the protein 2) Know the base sequence around the site to be altered Point mutation one base is altered A primer containing a mismatched nucleotide is used to produce a desired change in DNA sequence Insertions Cassette Mutagenesis a variety of mutations. Two restriction endonucleases are used to cleave plasmid, reintroduction of new DNA Designer genes create protein by splicing out gene segments then encode domains that are not associated in nature (Ex: Immnuotoxins and Synthetic Vaccines) Recombinant Methods Enable the Exploration of the Functional Effects of Disease Causing Mutations (Ex: Toxin SOD1 gene) Complete Genomes 1 bacterium H. influenza 1 eukaryotic Baker’s Yeast Human genome finished in 2004 Large number of pseudogenes formerly functional genes that no longer expressed Mobile genetic elements related to retroviruses, have inserted themselves throughout time Alu Sequences (~300 bases) Ex: SINES (Short Interspersed Elements) LINES (Long Interspersed Elements) Geneexpression levels can be comprehensively examined Quantitative PCR (qPCR) or “Real Time PCR” determine the quantity of individual mRNA transcripts by monitoring fluorescence (CT) is the threshold Transcriptome the pattern and level of expression of all genes in a particular cell tissue DNA Microarray fluorescently labeled cDNA is hybridized to the slide to reveal the expression level for each gene. Shows level of gene expression Introduction of Recombinant DNA Molecules into Animal Cells 1) DNA molecules precipitated by calcium phosphate are taken up by animal cells 2) Microinjection 3) Use of viruses to introduce new genes into animal cells (retroviruses most effective) Viral vectors: Proviral DNA using Moloney Murine Lukemia Virus as vector to introduce foreign genes in to mammalian cells Vaccinia virus Baculovirus Gene Disruption Provides Clues to Gene Function Gene disruption “Gene Knockout” Rely on process of homologous recombination Introduction of foreign mutated gene into an embryonic stem cell for recombination to take place RNA interference provides an additional tool for disrupting gene expression Tumorinducing plasmids (Ti Plasmid) Can deliver foreign genes into some plant cells Electroporation Introduction of foreign DNA into plant cells by application of the intense electric fields to make their plasma membranes transiently permeable Most effective: Gene gun Bombardmentmediated transformation Chapter 28 [Pg. 819846] DNA Replication, Repair, and Recombination 3 4 DNA synthesis (error rate 1 per 10 – 10 bases) Proofreading (reduce error rate 1 per 10 – 10 bp) Postreplication mismatch (reduces error rate 1 per 10 – 10 bp) DNA Replication Proceeds by the Polymerization of Deoxyribonucleoside Triphosphates Along a Template Each strand within the parent double helix acts as a template for the synthesis of a new DNA strand Building blocks deoxyribonucleoside triphosphates are added at the 3’end of the existing strand of DNA DNA Polymerase Require a Template and a Primer DNA Polymerases catalyze the formation of polynucleotide chains; template directed enzymes Need a primer with a free 3’hydroxyl group already baespaired to the template All DNA have Structural Features in Common Klenow fragment (of DNA polymerase I form E.Coli) Unit: fingers, thumb, palm, and a 3’5’ exonuclease activity hat participates in proof reading Two Bound Metal Ions Participate in Polymerase Reaction Usually Mg One metal ion binds both the deoxynucleoside triphosphate (dNTP) and the 3’ hydroxyl group of the primer, the other interacts only with the dNTP Two metal ions are bridged by the carboxylate groups of 2 aspartate residues in the palm domain to help stabilize the negative charge that accumulates on the pentacoordinate transition state The Specificity of Replication is Dictated by Complementarity of Shape Between Bases Shape Complementarity is important residues of the enzyme form hydrogen bonds with the minorgroove side of the base pair in the active site Shape selectivity the binding of dNTP to DNA polymerase induces a conformational change, generating a thigh pocket for the bp consisting of the dNTP and its partner on the template strand An RNA Primer Synthesized by Primase Enables DNA Synthesis to Begin RNA primes the synthesis of DNA RNA polymerase (primase) Synthesize a short stretch of RNA that is complementary to one of the template DNA strands After DNA synthesis has been initiated, the RNA is removed by hydrolysis and replaced by DNA One Strand of DNA is Made Continuously, Whereas the Other Strand is Synthesized in Fragments Replication fork Site of DNA Synthesis Okazaki fragments lagging strand synthesized in short pieces then rejoin by ligase Leading strand 5’3’ DNA Ligase Joins Ends of DNA in Duplex Regions DNA ligase catalyzes the formation of a phosphodiester bond between the 3’ hydroxyl group at the end of one DNA chain and the 5’phosphoryl group at the end of the other Eukaryotes and Archea ATP Bacteria NAD+ Ligase seals breaks in doublestranded DNA molecules The Separation of DNA Strands Requires Specific Helicases and ATP Hydrolysis Helicases Use ATP to power strand separation for replication 6 subunits each subunit has a Ploop NTPase domain 4 Nucleotidebinding sites each Only a single strand of DNA can fit through the center of ring; binds to loops on 2 adjacent subunits (one of which has bound ATP and the other of which has bound ADP+pi) DNA Unwinding and Supercoiling are Controlled by Topoisomerases The Linking Number of DNA, a topological Property, Determines the Degree of Supercoiling Linking number the number of times that a strand of DNA winds in the right handed direction around the helix axis when the axis lies in a plane Topological isomers (Topoisomers) molecules differing only in linking numbers; can be interconverted only by cutting one or both DNA strands and the rejoining them Twist Measure of the helical wining of the DNA strand around each other Writhe Measure of the coiling of axis of double helix (For unwound, W=0) Right handed coil is negative number Left handed coil is positive number L= W+T A lowering of L causes both right handed supercoiling of the DNA axis and unwinding of the duplex Topoisomerases Prepare the Double Helix for Unwinding Negative supercoiling is easier for strand separation than positive supercoiling Type I topoisomerase catalyze the relaxation of supercoiled DNA Type II topoisomerase use ATP to add negative supercoils to DNA Both alter the linking number of DNA by catalyzing 3 step processes: 1) Cleavage of one or both strands of DNA 2) Passage of a segment of DNA through this break 3) Resealing of DNA break Type I topoisomerases cleave one strand of DNA Type II topoisomerase cleave both strands Both use Tyr residue to form covalent links to the polynucleotide backbone Type I topoisomerases (in human) Has 4 domains Has Tyr 723 residue, which acts as a nucleophile to cleave the DNA backbone Tyr attack release a free 5’ hydroxyl group Type II topoisomerases to add negative supercoils Requires ATP Dimeric molecule Reaction begins with the binding of double helix Gsegment. Tyr residue of topoisomerase II form covalent linkage with the DNA backbone. Another double helix, TSegment comes in. ATP binds to the monomer of the enzyme leading to conformational change. Domains come close together, trapping the bound T Segment. The Tsegment passes through the cleaved GSegment. The ligation of Gsegment leads to the release of the Tsegment. Release of ADP opens up the domain. GSegment still bound This process decrease the linking by 2 [Bacterial] Topoisomerase II – “DNA Gyrase” DNA Replication is Highly Coordinated DNA replication requires highly processive polymerases Very high catalytic potency, fidelity, processivtity Processivity ability of an enzyme to catalyze many consecutive reactions without releasing its substrates DNA polymerase III dimeric beta subunit, beta subunit plays a key role in replication by serving as a sliding DNA clamp The Leading and Lagging Strands are Synthesized in a Coordinated Fashion Leading strand 1) DNA polymerase III begins the synthesis of the leading strand starting from the RNA primer formed by primase 2) The duplex DNA ahead of the polymerase is unwound by a hexameric helicase DnaB 3) Singlestrandedbinding protein (SSB) bind to the unwound strands, keeping them separated to serve as templates 4) Leading strand is synthesized by polymerase III 5) Topoisomerase II introduces negative supercoil to avert topological crisis DNA polymerase holoenzyme consists of 2 copies of the polymerase core enzyme Each core has α,ε,θ ,β2 subunits Linked to clamploader complex and helicase DnaB Clamploader is γτ2δδ ’ χ ,ϕ subunits interact with the SSB Lagging strand Trombone Model Formation of Okazaki fragments Lagging stand is looped out one at a time for formation of Okazaki fragment The gaps between fragments of the lagging strand are filled by DNA polymerase I, which uses 5’3’ enxonuclease activity to remove the RNA Primer DNA Replication in Escherichia Coli Begins at a Unique Site [In E.Coli] DNA replication starts at OriC locus which contains 5 copies of a sequence that are preferred binding sites for the origin recognition protein DnaA OriC locus contains a 13bp sequences that are rich in AT bp DnaA marks the origin of replication and favors DNA strand separation in AT rich sites 1) Binding of DnaA proteins to DNA is the first step in the preparation for replication. Binds to 5 high affinity sites 2) Single DNA strands are exposed in the prepriming complex AT rich site is separated into single DNA strands hold by SSB. DnaB helicase comes in Ready for synthesis of the RNA primers and assembly of the DNA polymerase II holoenzyme 3) The polymerase III holoenzyme assembles on the prepriming complex, triggers ATP hydrolysis within the DnaA subunits initiates DNA replication DNA Synthesis in Eukaryotes in Initiated at Multiple Sites Linear chromosomes are subject to shortening with each round of replication 1) The assembly of ORC (Origin of Replication Complexes)AT rich, is the first step in the preparation for replication 2) Licensing factors recruit a helicase that exposes single strand of DNA Cdc6 and Cdt1 proteins are recruited to recruit a helicase Mcm 27. Mcm 27 separates the parental DNA strands and single strands are hold by replication protein A (SSB) 3) 2 distinct polymerases are needed to copy a eukaryotic replicon Polymerase α has 2 subunits: primase and DNA polymerase is an initiator that begins replication, then is replaced by (polymerase switching) polymerase δ , which is the primary enzyme of DNA synthesis Replication Factor C (CRFC) displaces DNA polymerases α and attracks a sliding clamp proliferating cell nuclear antigen (pCNA). Binding of pCNA and polymerase δ long stretch of replication Checkpoints are required to control the progression Cyclin bind to cyclindependent protein kinases (CDK) and activate them to regulate replication Telomeres are Unique Structures at the Ends of Linear Chromosomes Telomeres Grich [AGGGTT] in human Protect end of chromosome Form a large duplex loop Replicated by telomerase Telomerase carries its own RNA template A specialized reverse transcriptase that carries its own template Errors Can Arise in DNA Replication Mutagenic can result in permanent changes in the DNA sequence Bases can be Damaged by Oxidizing Agents, Alkylating Agents, and Light Mutagens Ex: hydroxyl radical reacts with G to form 8oxoguanine, which pairs with A instead of C Ex: Deamination A is deaminated to form hypoxanthine pairs with C instead of T Ultraviolet light induces crosslinking between adjacent pyrimidines DNA Damage can be Detected and Repaired Proofreading ε subunit of E.Coli DNA polymerase II functions as a 3’5’ exonuclease that removes mismatch nucleotides from the 3’end Mismatch repair systems consist of at lease 2 proteins, one recognize and detect mismatch and other for recruiting an endonuclease to cleave the error site [E.Coli] MutS and MutL protein, MutH endonuclease Direct repair Ex: photochemical cleavage of pyrimidine dimers Photoreactivating enzyme – “DNA Photolyase” [E.Coli] DNA photolyase contains bound N , N – methenyltetrahydrofolate and FAD cofactors binds to the distorted region and uses light energy to form an excited state that cleaves the dimer into its component bases Baseexcision repair Ex: AlkA (missing purine base Apurine Site) binding of this enzyme to damaged DNA flips the affected base out inot the active site of the enzyme The enzyme acts as a glycosylase, cleaving the glycosidic bond to release the damaged base the missing base site is “AP site” (devoid of A or G) or (devoid of C or T) AP endonuclease recognizes the defect Deoxyribose phosphodiesterase excises the deoxyribose phosphate unit DNA polymerase I inserts an undamaged nucleotide sealed by ligase The Presence of Thymine Instead of Uracil in DNA Permits the Repair of Deaminated Cytosine Methylated base employ in DNA not in RNA because the methyl group on thymine is tag that distinguishes thymine from deaminated cytosine. Thymine is used instead of uracil in DNA to enhance the fidelity of the genetic message Some Genetic Diseases are Caused by the Expansion of Repeats of 3 Nucleotides Huntington Disease frequent repeat of trinucleotide CAG sequences Anticipation The children of the affect parents tend to show symptoms of the disease at an earlier age than did the parents Many Cancers are Caused by Defective Repairs of DNA Xeroderma pigmentosum (skin disease) mutation of excision repair pathway and Uvr ABC subunits HNPCC/ Lynch Syndrome Defective DNA mismatch repair mutation in hMSH2 and hMLH that code Muts and MutL P530 helps control damaged cells and promotes a DNA repair pathway or activated apoptosis Ames Test A petri plate containing ~10 Salmonella – “Salmonella test” A petri plate that contains bacteria that cannot synthesize His and a petri plate containing a chemical mutagen that produces revertant that can synthesize His To help evaluate the mutagenic and carcinogenic risks of a large number of chemicals DNA Recombination play Important Roles in Replication, Repair, and Other Processes Generate diversity RecA can Initiate Recombination by Promoting Strand Invasion The singleStranded DNA displaces one of the strands of the double helix 3 stranded structure (displacement loop D loop) – “Strand Invasion” Holliday Junctions Recombinase bind to the cross like structure and resolve them into separated DNA duplexes Recombination mechanism Begins as two DNA mocleules come together to form a recombination synapse. One strand from each duplex is cleaved by the recombinase enzyme (the 3’ end of each of the cleaved strands is linked to Tyr residue on the recomninase) New phosphodiester bonds are formed when a 5’ end of the other cleaved strand in the complex attacks the Tyr DNA adducts isomerization repeat Chapter 29 [Pg. 851882] RNA Synthesis and Processing RNA Synthesis Comprises 3 Stages: Initiation, Elongation, and Termination RNA synthesis is catalyzed by RNA Polymerases RNA Polymerases Catalyze Transcription Formation of a phosphodiester bond The 3’hydroxyl group of the last nucleotide in the chain nucelophilically attacks the α phosphoryl group of the incoming nucleoside triphosphate and release of a pyrophosphate Catalytic site of RNA polymerases 2 metal ions (normally Mg ) One binds to the enzyme, the other comes in with the nucleoside triphosphate and leaves with the pyrophosphate; 3 aspartate bind the metal ions Transcription Bubble Where polymerization reactions catalyzed by RNA polymerases take place RNA Chains are Formed De Novo and Grow in the 5’3’ Direction The coding strand of DNA has the same sequence as that of the RNA transcript except for T in place of U Coding strand= Sense (+) strand Template strand = Antisense () strand RNA can start De Novo without the requirement for a primer (DNA Can’t) Newly synthesized RNA tag on the 5’ end : pppG or pppA RNA Synthesis starts at the 5’end Elongation mechanism the 3’hydroxyl group at the end of the RNA chain attacks the newly bound nucleotide and forms a new phosphodiester bond, releasing pyrophosphate Translocation – after nucleotide addition, the RNADNA hybrid can translocate through the RNA polymerase, bringing a new DNA base into position to basepair with an incoming nucleoside triphosphate RNA polymerase forces the separation of the RNADNA hybrid RNA polymerases backtrack and Correct Errors Less favorable energetically breaks a bond by water attack the phosphate resulting in cleavage of the phosphodiester bond and release of the incorrect nucleotide For proofreading RNA Polymerase Binds to Promoter Sites on the DNA Template to Initiate Transcription Core enzyme – the bacterial RNA Polymerase with the composition α2ββ'ω Holoenzyme – additional subunit to the core enzyme σ2ββ'ωσ ; σ subunit helps find sites on DNA where transcription begins Promoter sites/ Promoters where transcription begins Two common motifs are present on the upstream side of the transcription start site; the 10 sequence and the 35 sequence Core promoter region containing the 10 sequence and 35 sequence 35: TTGACA 10: TATAAT Strong promoters promoter for this gene; transcribe every 2 secs Weak promoters transcribe every 10 mins – have multiple substitution at the site The efficiency / strength of a promoter sequence serves to regulate transcription Upstream element – a sequence of 4060 nucleotides upstream of the transcription start site Bound by α subunit of RNA polymerase and serves to increase the efficiency of transcription by creating an additional interaction site Sigma Subunits of RNA Polymerase Recognize Promoter Sites α2ββ'ω core of RNA polymerase must bind to promoter to initiate transcription σ subunit makes the binding possible by enabling RNA polymerase to recognize the promoter site – acts catalytically σ32 Recognizes the promoters of heatshock genes RNA Polymerase Must Unwind the Template Double Helix For Transcription to Take Place DNA unwinding the transition from the closed promoter complex requires the unwinding of approx. 17 bp of DNA Elongation Takes Place at Transcription Bubbles that Move Along the DNA Template Duplex DNA is unwound at he forward end of RNA polymerase and rewound at its rear end. The RNADNA hybrid rotates during elongation Sequences Within the Newly Transcribed RNA Signal Termination The transcribed regions of DNA template contain stop signals Ex: palindromic GCrich region followed by an ATRich region forms hairpin followed by addition of 4 or more Uracil residues Dissociation Some Messenger RNAs Directly Sense Metabolite Concentrations As Control Mechanism Riboswitches mRNA molecules that can directly bind to small molecules The Rho Protein Helps to Terminate the Transcription of Some Genes Rho (p) – provoke termination of RNA synthesis Hydrolyzes the ATP in the Presence of Single Stranded RNA but not in the presence of DNA or duplex RNA Rho catches the RNA polymerase at the transcription bubble; it breaks the RNA DNA hybrid helix by functioning as a RNADNA helicase Another Ex: NusA Protein Functioning signals lie in newly synthesis RNA rather than the DNA template Some Antibiotics Inhibit Transcription Rifampicin and Actinomycin inhibit bacterial transcription Rifampicin inhibits the initiation of RNA synthesis Actnomycin binds tightly and specifically to doublehelical DNA and prevent it from being an effective template for RNA synthesis (Intercalation) Precursor of Transfer and Ribosomal RNA are Cleaved and Chemically Modified After Transcription in Prokaryotes Transfer RNA and ribosomal RNA molecules are generated by cleavage and other modifications of nascent RNA chains 1) Nucleases cleave and trim the precursor of rRNA and tRNA 2) Addition of nucleotides to the termini of some RNA chains 3) Modification of bases and ribose units of ribosomal RNAs Allow greater structural and functional versatility Transcription in Eukaryotes in Highly Regulated Multicellular eukaryotes use differential transcriptional regulation to create different cell types 1) The nuclear membrane. In eukaryotes, the transcription and translation take place in different cellular compartments Transcription in nucleus Translation in cytoplasm 2) Complex transcriptional regulation [In eukaryotes] elements that regulate transcription can be found at a variety of locations in DNA 3) RNA processing eukaryotes very extensively process nascent RNA destined to become mRNA Types of RNA polymerase synthesize RNA in Eukaryotic Cells RNA polymerase I in nucleoli RNA polymerase III – in nucleoplasm, synthesize tRNA RNA polymerase II in nucleoplasm, synthesize the precursors of messenger RNA and precursors and small regulating RNAs Contains unique carboxylterminal domain (CTP)= repeats of YSPTSPS RNA polymerase I promoter at the upstream promoter element TATA like sequence “ribosomal initiator element (rInr) next to it Transcribe rDNA RNA polymerase II promoter may include: Inr, DPE, or TATA box RNA polymerase III promoter promoters are within the transcribed sequence, down stream of the start site Type I: 5s rRNA, contains A block and C block Type II: tRNA, contains A block and B block 3 Common Elements can be Found in the RNA Polymerase II promoter Region 1) Cisacting element – generally on the 5’ end Most common TATA box 2) Initiator element (Inr) – defines start site, often paired with TATA box 3) Downstream core promoter element (DPE) CAAT box 5’ GGNCAATCT 3’ GC box 5’ GGGGGG 3’ The TFID Protein Complex Initiates the Assembly of the Active Transcription Complex Transcription factors are required TFII: TFIIA, TFIIB….Essential in initiating transcription by RNA polymerase II. Binding of TFIID to TBP (TATA box binding protein), TFIID then opens the DNA double helix and phosphorylates the CTD begins transcription Hydrophobic interaction at the surface TBP bound to the TATA box is the heart of the initiation complex Basal Transcription Apparatus binding of TFIIA, TFIIF, Polymerase II, TFIIE, and TFIIH Phosphorylation of the CTD by TFIIH marks the transition from initiation to elongation Multiple Transcription Factors Interact with Eukaryotic Promoters Heatshock transcription factor (HSTF) in Drosophila Upstream of TATA box Enhancer Sequences can Stimulate Transcription at Start Sites Thousands of Bases Away Enhancer a cisacting element Have no promoter activity of their own but can exert their stimulatory actions over distance of several thousand base pairs A particular enhancer is effective only in certain cells Transcription factors and other proteins that bind to regulatory sites on DNA can be regarded as passwords The Transcription Products of Eukaryotic Polymerase are Processed Nearly all mRNA precursors in higher eukaryotes are spliced: introns are spliced and exons are joined to form mature mRNA RNA Polymerase I Produces 3 Ribosomal RNAs RNA polymerase I makes: 18S rRNA, 28S rRNA, and 5.8S rRNA Processing eukaryotic prerRNA is directed by SnoRNPs (small nuclear ribonucleoproteins) Nucleotides are modified: SnoRNPs methylate specific ribose groups and convert selected uredines into pseudouridines PrerRNA is cleaved and packaged to form mature ribosomes RNA Polymerase III Produces Transfer RNA tRNA transcripts are among the most processed of all RNA polymerase III transcripts The Product of RNA Polymerase II, the PremRNA Transcript, Acquires a 5’ cap and a 3’ Poly A Tail Immediate product of RNA polymerase II is : PremRNA Modification of premRNA 5’ cap 7 methylguanylate attached by a triphosphate linkage to the ribose ; 0 methylated ribose = cap 0, 1 methylated = cap 1, 2 methylated= cap 2 Cap protects the 5’ end from phosphatase and nucleases 3’end poly A tail, added after transcription has ended Eukaryotic primary transcripts are cleaved by a specific endonuclease that recognizes the sequence AAUAAA Small Regulatory RNAs are Cleaved from Larger Precursors MicroRNAs plays a role in gene regulations in eukaryotes Fold into hairpin that are cleaved by specific nucleases Mature microRNA is bound by Argonaute RNA Editing Changes the Protein Encoded by mRNA RNA editing a change in the nucleotide sequence of RNA after transcription processes other than RNA splicing Apolipoprotein (apoB) plays a role in the transport of triacylglycerols and cholesterol by forming an amphipathic spherical shell around lipids carried in lipoprotein particles 512 kd apo B100 – synthesized by liver 240 kd apo B48 – Synthesized by small intestine Changing of the nucleotide sequence of mRNA after its synthesis to generate diversity A specific cytidine residue of mRNA is deaminated to uridine, which changes the codon at residue 2153 from CAA (Gln) to UAA (stop) The enzyme for this is only in the small intestine Sequences at the Ends of Introns Specify Splice Sites in mRNA Precursors The introns begins with GU and ends with AG 3’ end of intron has a stretch of 10 pyrimidines (U or G) = Polypyrimidine tract Branch site Between 20 and 30 nucleotides upstream of 3’ splice site 5’ and 3’ splice sites and branch site essential for determining where splicing takes place Splicing Consists of Two Sequential Transesterification Reaction Spliceosome splicing begins with the cleavage of the phosphodiester bond between the upstream exon (exon 1) and the 5’ end of the intron. The attacking group is the 2’OH group of the adenylate residue in the branch site Transesterification Lariat intermediate when the 5’ splice site is bonded with the branch site and the 3’ OH of Exon 1 is attacking Exon 2 Splicing is accomplished by 2 transesterification reaction – the number of phosphodiester bonds stays the same during these steps Result: spliced product and a Lariat form of intron (bound by U2, U5, U6) Small Nuclear RNAs in Spliceosomes Catalyze the Splicing of mRNA Precursors SnRNAs: U1, U2, U4, U5, U6 SnRNPs “Snurps” [Mammalian Cells] splicing begins with the recognition of the 5’ splice site by the U1 SnRNP U1= Binds the 5’ splice site U2= Binds the branch site and part catalytic center U5= Binds the 5’ splice site and then the 3’ splice site U4= Inhibitor to mask U6 U6= Catalyzes Splicing Psoralen a reagent that joins neighboring pyrimidines in bp regions on treatment with light Transcription and Processing mRNA are coupled CTD contribution to efficient transcription by: 1) Capping enzymes, methylate the 5’ G on the PremRNA 2) Components of the splicing machinery, initiate the excision of each intron 3) An endonuclease that cleaves the transcript at Poly A Tail. Free 3’ OH to target for 3’ adenylation CTP = Carboxyl terminal domain of polymerase II Most Human PremRNAs can be Spliced in Alternative Ways to Yield Different Proteins Alternative Splicing Creates diversity The Discovery of Catalytic RNA was Revealing in Regard to Both Mechanism and Evolution Ribozyme RNA that can splice itself G is required a cofactor in GMP, GDP, or GTP, which attacks the 5’ splice site to form a phosphordiester bond with 5’ of intron Has Gbinding site Splice sites are aligned with internal guide sequence (IGS) catalytic residues in the intron and the 5’ and 3’ exons [Tetrahymena] Group I self splicing is mediated by a G cofactor Group II splicing is the attacking of 2’ OH group of a specific adenylate of the intron
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