MAD seminar tonight CF 110 5:15 pm
Crystal structures of AlkD in complex with 3d3mA-DNA (a) and THF-DNA (b). EH Rubinson et al.Nature000, 1-6 (2010) doi:10.1038/nature09428 The modified 3d3mA and tetrahydrofuran (THF) nucleotides are colored blue, and the opposing thymine is magenta.
DNA positioned along +charged concave surface of alkD Contacts 10 bp Contacts cluster around mismatch Fewer contacts on lesion strand
Both 3m3A and THF reside on the face of the DNA duplex NOT in contact with the protein whereas the compelement is nestled in the enzyme cleft. This THF trapped complex shows the abasic site rotated 90o around the PD backbone where it is totally solvent exposed!! Opposing T has slipped out of helix into DNA minor groove!! Backbone distorted NO aa PLUG DNA duplex has collapsed to retain stacking interactions.
Remodelling of a G•T wobble base pair by AlkD. a, AlkD–G•T-DNA complex viewed down the helical axis. b, The structure of a G•T wobble base pair in DNA alone is superimposed onto the AlkD–G•T complex. Steric clashes between the protein and DNA are highlighted by yellow stars, and disrupted hydrogen bonds (dashed lines) are shown by a red X. c, Relative single-turnover rates (kst) of 7mG excision from a 25mer oligonucleotide duplex by wild-type AlkD and the indicated AlkD mutants. AlkD seems to detect DNA duplex destabilization rather than specifically recognizing modified bases. Protein restructures the wobble and disrupts base stacking.
Solvent exposure increases the lifetime that spontaneous depurination is likely to occur!!!The phosphate groups on the DNA may participate in the rate enhancement by positioning water molecules for solvent attack on the glycoside bond.
AlkD captures the DNA in an orientation that holds the orphaned base next to the protein and exposes the lesion to a hydrolytic environment. The distorted DNA conformation is stabilized not by a side chain plug, but by stacking of flanking base pairs as a result of both lesion and orphaned base flipping. Phosphate (gold “P”) assisted hydrolysis could occur either by positioning of the water molecules adjacent to the C1' carbon in a dissociative hydrolysis reaction, or through stabilization of an oxocarbenium ion intermediate Figure S12. Proposed mechanism for how AlkD facilitates hydrolysis of N3 and N7-alkylpurines by distorting the DNA backbone.
MISMATCH REPAIR: only viable a short time after replication Methylation and mismatch repair Really only understood well in E.coli. The methylation occurs at the N6 of adenines in (5′)GATC sequences. (palindrome) Dam=DNA adenine methylation
Double strand repair • Homologous end-joining • damaged site is copied from the other chromosome by special recombination proteins
Double strand repair • Nonhomologous end-joining • only in emergency situations • two broken ends of DNA are joined together • a couple of nucleotides are cut from both of the strands • ligase joins the strands together
Cell Cycle and DNA repair • Cell cycle is delayed if there is a lot of DNA damage. • Repairing DNA as well as signals sent by damaged DNA delays progression of cell cycle. This ensures that DNA damages are repaired before the cell divides
Overview of RNA Function Ribonucleic acids play three well-understood roles in living cells Messenger RNAs encode the amino acid sequences of all the polypeptides found in the cell Transfer RNAs match specific amino acids to triplet codons in mRNA during protein synthesis Ribosomal RNAs are the constituents and catalytic appropriate amino acid Ribonucleic acids play several less-understood functions in eukaryotic cells Micro RNA appears to regulate the expression of genes, possibly via binding to specific nucleotide sequences Ribonucleic acids act as genomic material in viruses
Replication vs. Transcription Both add nucleotides via an attack of the 3’ hydroxyl of the growing chain to -phosphorus of nucleoside triphosphates RNA synthesis requires ribonucleoside triphosphates RNA synthesis pairs A with U instead of dA with dT Both require catalysis by a Mg++-dependent enzyme RNA synthesis has lower fidelity RNA synthesis does not require a primer for initiation Both require a single strand of DNA as molecular template for building the new strand
Adenovirus: Many serotypes that mostly cause respiratory infections, especially in crowded situations. Some new studies link Adenoviruses with some types of human obesity! Organization of coding information in the adenovirus genome.
Hexons and pentons form capsid (TP) Covalently linked to DNA 36 kbp 50 nm Transcribed by RNA pol II Transcribed by RNA pol III Figure A-1 Adenovirus
(prok) Inh. initiation Euk inhibitor Blocks elongation MVA Fig. 26.4
The shaded portion of actinomycin D is planar and intercalates between two successive G≡C base pairs in duplex DNA.
Bacterial RNA Polymerase has at Least Six Subunits Two two subunits function in assembly and binding to UP elements The subunit is the main catalytic subunit The ’ subunit is responsible for DNA-binding The subunit directs enzyme to the promoter The appears to protect the polymerase from denaturation
X-Ray structure of Taq RNAP core enzyme. a subunits are yellow and green, b subunit is cyan, b¢ subunit is pink, w subunit is gray. What do you notice about this structure?
The Subunit Binds to Promoter Sequences in DNA • Typical promoters have TATA sequence about 10 base pairs before the transcription starts side • AT-rich sequences promote strand separation during initiation
Footprint analysis of the RNA polymerase-binding site on a DNA fragment. Separate experiments are carried out in the presence (+) and absence (–) of the polymerase.
The sense (nontemplate) strand sequences of selected E. coli promoters. Page 1223
The sequence of a fork-junction promoter DNA fragment. Numbers are relative to the transcription start site, +1.
Model of the open (Rpo) complex of Taq RNAP with promoter-containing DNA showing the transcription bubble and the active site. Page 1225
Figure 31-14 The two possible modes of RNA chain growth. Growth may occur (a) by the addition of nucleotides to the 3¢ end and (b) by the addition of nucleotides to the 5¢ end. Page 1226 How could you distinguish between these two possibilities?
Figure 31-16 An electron micrograph of three contiguous ribosomal genes from oocytes of the salamander Pleurodeles waltl undergoing transcription. Page 1228
Transcription Initiation and Elongation in E. coli In the closed complex, the DNA in the promoter region is bound to polymerase but not unwound In the open complex, the two chains in the AT-rich promoter region region are separated subunit leaves before elongation starts
RNA Backtracking MVA Fig. 26.10
-Independent Termination of Transcription in Prokaryotes The RNA polymerase pauses at certain sequences during transcription Some sequences allow formation of the hairpin within the product If the polymerase pauses for long enough and the hairpin forms, the RNA-DNA hybrid is disrupted This promotes dissociation of the polymerase
Eukaryotes Contain Several Distinct Polymerases RNA polymerase I synthesizes pre-ribosomal RNA (precursor for 28S, 18S, and 5.8 rRNAs) RNA polymerase II is responsible for synthesis of mRNA Very fast (500 – 1000 nucleotides / sec) Specifically inhibited by mushroom toxin -amanitin RNA polymerase III makes tRNAs and some small RNA products Plants appear to have RNA polymerase IV that is responsible for the synthesis of small interfering RNAs Mitochondria have their own RNA polymerase
Formation of Primary Transcript and Its Processing in Eukaryotes • The removal of introns from the primary transcript is called splicing
RNA Processing Almost all newly synthesized RNA molecules (primary transcripts) are processed to some degree in eukaryotic cells The 5’-end is capped w/ methylguanosine Introns are spliced out Poly-A tail is built at the 3’ end Processing is catalyzed by protein-based enzymes and by RNA-based enzymes (ribozymes) Only some prokaryotes have to splice out introns but many process their tRNA precursors
Capping the 5’ of mRNA • Capping protects mRNA from 5’exonuclease degradation