CH07_HO - Major differences between prokaryotic and...

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Unformatted text preview: Major differences between prokaryotic and eukaryotic DNA Bacterial DNA Eukaryotic DNA Template Exposed Masked {condensed chromatin} Default state Available for transcription Unavailable for transcription Regulation emphasis Negative (repressors). Activation. to overcome an to counter accessibility inactive template MORE RECENT DNA FOOTPRINTING (1994} A method to establish where on DNA a protein binds or whether the protein is present Uses a single DNA stretch produced by PCR DNA Fragment non-DNA-bindin “We “MM DNA Bindin Fmiein 5”” Protein 9 E-carbumiuorescein 5' ' lillilillliilliliil Endm‘uc'fl“ liliilililltiiiltllll Digestion [Dflase I) Capl Ilary Electrophoresis ml fig 0 ml 0M 0 Size (lap) Size (hp) I‘fu: relative fuorescence units. In capillary electrophoresis a smail capiliary tube tilted with an eiectrolyte is used to separate moiecules based on their size—to—charge ratios mm... I’“l.......... mm... 1 Digested samples undigested FOOTPRINTING 1| n I | l lllllllllllyfl i'll'lllll L. l iiili li llllll - l 4 Labeled DNA DNAse | —-“—- amt? imam it}. (0:5 I lmmflm angimnmmrtmllidml Imam Ill lllllll‘ mum Illl III II . lllll ll mm ‘ m "'l l .E 'llll'fl lll Ill .: Ill r ll Ill l Ill lIIIl Illl GAlll II I Tallulll ‘ illll Onlythe labeled DNA segments can be visualized. Lanes "GA" and "To. are chemical sequencing Ianea § 2 g g a. Gene transcription is a controllable process 2 F g protein {:1 M } unprotected Positive control: low levels of a metabolite may induce transcription of genes involved in producing that metabolite from precursors. Negative control: repressors inhibit transcription when bound to the DNA template. Transcriptional attenuation: regulation of the expression of genes that control RNA polymerase activity. Cis-acting control: regulation of downstream genes. Trans-acting control: regulation by DNA-associated molecul interact with DNA of other chromosomes. Roger Kornberg’s model of the Yeast RNAPII - The two largest subunits (prl and pr2) define a deep cleft through which the DNA template enters. 2006 Nobel Prize (chemistry) (father, Med 1959 Math of gene transcr) \. k »\ . fl h- l . . - Other. smaller subunits are arranged around these two prs. R b1 and R b2 are the Iar est and second Iar est subunits of euka otic RNA p p g Pol II. respectivei, W RNA polymerase (T?) complexed to DNA and RNA RNA polymerase is a nucleotidyl transferase that polymerizes ribonucleotides at the 3’ end of an RNA transcript Products of RNA polymerase include: mRNA: the template for translation. RNA genes: non-coding genes (encoding RNA that is not translated into protein. E.g.. tRNA and rRNA. both involved in translation). Micro RNA: regulates gene activity Ribozymes: enzymatic RNA RNAP Eukaryotes have several types of RNA polymerase characterized by the type of RNA they synthesize. iflSCr’lpthfl Video: http waw youtube comfwatch?v=rRoqfoLuu00 KorZheVa. N. eta! Science 2892 619. 2000 Roger Kornberg’s model of MEDIATOR I TERMINATION OF TRANSCRIPTION BY RNA Pol l is a two-step process 1) pausing of the transcription-elongating complex and 2) Dissociation: separation of Pol l and pre—rRNA from the DNA template In the mouse, pausing is mediated by TTF1 (transcription termination factor 1) bound to a Sal box terminator (these are 18-bp-Iong DNA elements that contain several San restriction sites - 5’ G'TCGAC 3’). A ribosomal DNA gene could have as many as 8 Sal boxes at its 3’ end). Inverting the sequence abrogates termination. Dissociation (release) of the paused Pol | complex requires PTRF (Pol | and transcript release factor), which helps dissassemble the Pol | ternary complex . MISCELLANEOUS: Termination of transcriptioanNA synthesis Transcription by RNA Pol II is not really terminated at the end of a gene in the sense that transcription continues for LOGO-2,000 bases downstream from the site that ultimately will become the 3' end of the mature transcript. The “functional transcript actually results from subsequent cleavage of the primary transcript (RNA processing). Termination of transcription via RNA Poll and RNA Pol III is determined by discrete termination signals. For Pol l: several Sal boxes (5' G'TCGAC 3') For Pol III: short stretch of T (TTTT) Transcription enhancers and silencers: these are sequences upstream or downstream of the transcribed sequence that enhance and reduce, respectively, rates of transcription. Most enhancers and silencers are not well characterized and can occur close to or distant (>1,000 bp) from the gene(s) they affect. Polyadenylation: an enzyme [poly(A) polymerase] adds about 200 adenine (A) bases to the 3' end. This is called polyadenylation or addition of the poly-A tail. The functions of poly-A tails added to mRNA are to increase the stability of the transcript and assist in transporting mRNA from the nucleus to the cytoplasm. This is another aspect of RNA processing in eukaryotes. The poly-A tail also plays a major role in initiating translation. Binding of regulatory factors to DNA isjust a chemical reaction HELIX-TURN-HELIX Bicoid sire. base pair 7 Antp site, base pair 7' {Tylminc (iuaninl: Thyminc Adenine ” f”? (kc/K CH, / \CH CH. CH/ 3 \(‘H 1\ 1-.— Antp \ /‘ 3 “.3 Bicoid fist; TF TF "911% Left: proposed pairing between the lysine of the Bicoid homeodomain and the (3:6: base pair of its recognition sequence. Right: pairing between the glutamine of the Antennapedia (Antp) homeodomain and the T:A base pair of its recognition sequence. Bicoid and Antp are Drosopnila proteins. DNA H1 h' t 15 homo.o;:::l:ie:nns, Nucleosome Linker H4 103 M 11 7 The unit of chromatin is the nucleosome‘ consisting of 146 bp of DNA wound in nearly two turns around a disk—like protein octamer consisting of histones. The core is an octamer made of 2 H3 and 2 H4 complexed with 2 H2A and 2 HZB, although some variant forms also exist. such as H2A.Z. Histones are rich in the positively charged amino acids Lys and Arg, and thus have affinity for the negatively charged phosphate backbone of DNA. H1 phosphorylation relaxes DNA; dephosphorylation compacts DNA. EFFECTS OF HISTONE ACETYLATION ON CHROMATIN CONDENSATION DEACETYLATED NUCLEOSOME ACETYLATED NUCLEOSOME condensed (inactive) decondensed (active) Amino-terminal tail domains _ NOMENCLATURE FOR HIVIG PROTEINS / \ HMGsBox DNAsblfldlflg protein HMG Motif Canonical ofD meianogasier proteins HMG Proteins Func'ii onal M olif _ HMG-Elox HMG-Box proteins W New Name: HMG Box _ _ _ _ . . oo sym 0 Old Name HMG-Ii‘Z Contaln1 or 2 30 a.a. DNA binding HMGB “akin”chmmiwmdmmflulm motifs (boxes) that specifically or non-specifically bind dsDNA DNA-dependent activities HMG Box protein NUCIEOSDI‘I‘IG binding 4—h- I I I I I I I I I I I I I I . . I I I I : Rootsiirnboi' : Old Name HMGVMJVIT I I I I I I I I I I I I . . I I I I DNA - NED-proteins New Name; HMGN l Typically contain a HMGN destabilize chromatin nucleosome-binding domain fl AT-hook . AT-hook proteins 4—" New Name: HMGA x \ x R t b l ’ \' Typically contain 2 or 3 AT hooks in;ng O'd_'_“3’"e WG'W (P-R-G-R-P sequences). which bind mh'h" ATmh DNA minor groove runs of NT bases — — — — — — — — — — — — — The functional motifs characteristic of each of the canonical high-mobility group (HMG) proteins are widespread among nuclear proteins. Proteins containing any of the functional motifs of canonical HMG proteins are now called HMG-motif proteins. The HMG motif proteins are now subdivided into 3 THE HMGN PROTEIN FAMILY NBD PROTEINS superfamilies: HMGB, HMGN, and HMGA. Each HMG family has a characteristic _ _ functional sequence motif: “HMG-box”, “nucleosomal-binding domain,” and NLS - - g‘V‘T-hook,” respectively. NBD "Ls UNFOI‘DING —5l HMGN1 || Mammalian examples Human HMGNSa HMGN3a II as: 33333” Mouse HMGNSa HMGN3b IWI Mouse HMGN3b HMGN4 IMI Members of the HMGN family (NBD) of proteins contain 3 functional domains: NLS: a bipartite nuclear localization signal NBD: a nucleosomal-binding domain Unfolding: a chromatin-unfolding domain HMGN proteins are the only non-histones known to bind specifically to the nucleosome DNA- histones complex (not to the linker). For example. HMGN1 binds so that its 0- and N-terminal regions are located near the N-terminal tails of H3 and H23. respectively: additional HMGN- histone contacts have also been identified. HMGN-nucleosome interaction facilitates HAT-mediated acetylation of H3 Lys14. reduces chromatin compaction. favors accessibility of unique sites in the nucleosome to other proteins. and enhances the rate of transcriptional initiation. HMGA - AT-hook binding to DNA - HMGA proteins possess 3 similar, but independent, AT hooks which have an invariant consensus (PRGRP) motif flanked on either side by positively charged residues. - HMGA proteins bind‘ via their AT hooks, to stretches of AT- rich DNA in the minor groove but seem to recognize substrate structure rather than nucleotide SEQUENCE. Key facts about tRNA 0 Each tRNA molecule has a specific anticodon o Each tRNA molecule has a specific amino acid-acceptor site 0 Each tRNA molecule has a specific “charger protein" (aminoacyi IRMA synthetaae) o The energy used to bond the amino acid to the tRNA is provided by ATP 0 The first tRNA engaged in peptide synthesis has an anticodon for AUG (Met) THE WOBBLE POSITION IN tRNA Immediately after synthesis, tRNAs - like all RNAs - contain only A, G, C and U. However, mature tRNAs contain modified bases, i.e., bases other than these four. These modifications are introduced after transcription of the tRNA-encoding genes. lVlost modified bases occur at restricted sites in the tRNA molecule. However, occasionally, the first base (5‘) of an anticodon, the wobble position, is modified and allows the formation of unusual base pairs with the 3' base of the mRNA codon. These tRNAs are functional. 3‘} o inosme Processing protein-encoding eukaryotic RNA 8.3.3 equivalent Prlmary 5: UTR Exon 1 Intron 1 Exon 2 ‘ Intron 2 EXOI] 3 3’ UTR transcript Processing (splicing) 3, mRNA S’UTR Exonl Ex0112 Ex0113 3'UTR AAAAAAAAAA... A 5’ Cap 3’ Poly A tail Splicing pre-mRNA processing achieves three things The addition of a 5‘ cap The removal of introns by splicing The addition of a 3‘ poly-A tail This signals that the mRNA is ready to move out ofthe nucleus. The life span of mRNA is controlled in the cytoplasm. After cleavage, PAP creates the poly(A) tail. ‘5 - AAAMM Then, PAP is signaled to stop adding A residues. I PAEII A short tail is slowly created. Then, additional proteins accelerate the process. EB 1" Processing is complete and the molecule is ready for splicing. PAP: poly(A) polymerase mRNA SPLICING “‘i ‘1“ U2 InRHA U‘l snRNA Excm1 \ / Exon 1 Exon2 lI-un 1 Formation of spliceosomes in mammalian cell nuclei. (A) Spliceosomes bound to a fragment of human pre-mRNA. (B-C-D) Model of spliceosome formation based on the progressive binding of snRNPs to the branch point and splice sites of the pre-mRNA. (E) Lariat formation and splicing. ALTERNATIVE pre-RNA SPLICING Production of membrane and secreted antibodies Transmembrane lgM L VDJ 0M1 cuz CHE 0,14 30 pAS MC pa... D“ it.— = "n+- = Trmripflm Prim! . . . . . . - [I - --- 1am Secreted .9.“ “mt Cieava a at second ' ponAgdclitiun sile L. VDJ CHI 0P2 CPS 0P4 SC MS MO pAm (pi-n: am spicung . Rearranged DNA slim—41+: -—l— I]- W “3mm Translation. Prlma pruieln r ,r transcript in {i processing I v “A u 21. IO" 5‘ H I u msmemrm ‘9“ FE: I and splicing ’ Ii mRNA --l_- W Translation. pmiain Jaheway's lmmunobiology, Garland Publ fl mom“ C iarrniniis RNA EDITING Replaces Cooper & Hausman ’s 7.52 Occasionally genes are identified whose nucleotide sequence is not matched exactly in its RNA product (mRNA, rRNA or tRNA). RNA editing occurs AFTER transcription. SUBSTITUTION EDITING is a chemical alteration of individual nucleotides (equivalent of point mutations). These alterations are catalyzed by enzymes that recognize a specific target sequence of nucleotides. c idine deaminases yt—r A adenosine deaminases I . . . . —P . . . . .. INSERTIONIDELETION EDITING results in the insertion or removal of a nucleotide in preRNA. It is mediated by guide RNA molecules that base— pair as best they can with the RNA to be edited and serve as a template for the addition or removal of nucleotides in the target RNA. INSERTIONIDELETION (INDEL) EDITING lys 1l'al GIJ Asn Leu Val DNA S’...AAAGTAGAEAA_EETGGTAGE...3" ¢ Transcription INDEL GuideRNA.:-'...UUAUAUEUAAUAUAUEGAUAAUAU In, _. HHIIIHHHHIIIHHH "Semon Pre-mRNAS...AAAGUAGA G A ACCUEGUAGG Deletion (not shown) * Insertion editing editing EditedmRNAE" ...AAA|3 U A G A U U G U A U AC E U E G U AG [3... 22:" $ Translation of edited RNA ...
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CH07_HO - Major differences between prokaryotic and...

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