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DNA, RNA polymerase, Mating type switching of S. cerevisiae;, Requires ATP…

- - - - Presence of another undamaged and homologous strand is necessary. Therefore, homologous recombination can only occur during cell cycle phases in which a sister chromatid or chromosomal copy is present
        
        5'-ends are degraded to create a 3' overhangs and 2-strand invasions by the intact strand via recombinases create a D-loop, which allows the damaged DNA to extend its strands via DNA polymerase as ligation.
        
        Double-Strand Break Repair
        
        Holiday intermediates formed; Cleaved via holiday junction resolvases and filled via DNA ligation
        
        YxY, XxX cuts
        
        Non-crossover products (for each chromatid, one strand homo- other hetero-)
        
        XxY, XxY cuts
        
        Crossover products (for each chromatid, both strands hetero-)
        
        Synthesis-Dependent strand annealing (SDSA)
        
        (Most common!) Strand dissociation and annealing to original sister strands, completion of replication and ligation
      - Site specific (ch 14)
        
        Recombination sites recognized by site-specific recombinases are two inverted repeats separated by an asymmetrical core sequence.
        
        Recombination of two sites with same orientation
        
        Insertion/Deletion
        
        Recomb w sites of opposite orientation
        
        Inversion of sequence
        
        Recombination can only occur if the sequences of the two cores are identical and proceed in the same direction
        
        Viral infection cycle (Ch 20)
        
        Lytic cycle: Phage DNA is replicated and packaged into new viral heads and host cell is lysed for dispersement
        
        Cro predominates
        
        Binds to Or3 to turn off Prm and inhibit c1 to enter lytic phase
        
        Lysogenic cycle: Prophage is inserted into host cell for passive replication
        
        lambda repressor (c1) predominates
        
        Binds Or1 and Or2 to suppress Cro and enter lysogenic cycle
        
        c2 activates transcription of lambda proteins; too susceptible to proteases, so not usual; c3 stabilizes
        
        Key nucleophiles in the active site
        
        Tyr
        
        2/4 recombinases use Tyr to attack phosphodiester bond
        
        2 separate reactions occurring to complete recombination
        
        Dimeric>monomeric chromosomes in E. Coli
        
        Uses XerCD recombinase
        
        ser
        
        Both strands cut concurrently and rejoined to new partners WITHOUT holliday intermediate
        
        All 4 recombinases participate, so ser recombinases only use 1 reaction
        
        Transposition
        
        Transposons jump from the donor site (one place on the chromosome) to the target site (another place on the chromosome)
        
        Transposable systems composed of transposon and the enzyme transposase
        
        Transposase encoded for within the transposon
        
        Catalyzes hydrolysis of phosphodiester bond at the end of the transposon and attack of a freed 3'-OH on another phosphodiester bond (transesterification)
        
        Normally leaves gaps in DNA, filled by DNA repair mech. (DNA pol and ligase)
        
        DNA sequence homology is not required; new sites are chosen randomly, but most transposons have a degree of target specificity
        
        Cut/Paste
        
        Excised via ds cuts on each side and transposon moves to new location
        
        Hairpin int. before addition to new strand cleaved by transposase
        
        Retrotransposons
        
        Non-LTR (target primed)
        
        LINEs
        
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        SINEs
        
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        Migrate via cut/paste (Tc1/mariner)
        
        Replicative
        
        Transposon is not completely excised before beginning transfer on new strand; one stand from each chromosome is joined with another strand of the target forming an intermediate
        
        Free 3' ends used as primers, and transposon section is replicated= cointegrate
        
        Cointegrate resolved via homologous recombination with two copies of the transposon; one in donor, and one in target
        
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        RNA intermediate
        
        Retrotransposons: Long terminal repeat (LTR) sequences at termini
        
        Reverse transcriptase can use either DNA or RNA as a template; uses RNA to encode a complimentary DNA strand
        
        Bacterial have three classes of transposons
        
        Insertion sequences
        
        simple transposons with only the sequences required for transposition
        
        Composite transposons
        
        Contain 1+ genes in addition to those necessary for transposition
        
        Some extra genes may contribute to the survival of the host cell (Ex. antibiotic resistance)
        
        usually 2 insertion sequences flanking other genes
        
        Ex. Tn5 have two IS elements flanking genes that help in antibiotic resistance (kanamycin, bleomycin, streptomycin)
        
        Complex transposons
        
        Larger genomes; include genes for auxillary proteins that assist transposase/ promote other processes
    - - RecA
        
        RecA: DNA dependent ATPase used to promote DNA strand exchange
        
        RecA first blocked by SSB proteins; Loaded onto ssDNA by recFOR for ssbreaks and recBCD in ds breaks
        
        SOS response usage
        
        When RecA binds ssDNA breaks, LexA is cleaved, allowing transcription of SOS genes
        
        Regulation of RecA
        
        A mutant RecA can displace SSB proteins without the need of RecFOR recruitment, leading to higher levels of recombination
        
        RecX binds to growing RecA filament, halting extension
        
        DinI binds to RecA filament to stabilize it and limit DNA branch migration
        
        UvrD (helicase) removes RecA filaments when not needed
      - RecFOR
        
        RecFOR used to displace SSB to allow RecA to bind ssDNA
      - RecB
        
        Helicase motor running 3' to 5'
        
        Has nuclease domain that degrades both strands at an equal rate until reaching chi sequence, then nuclease only continues to degrade 5'-ending strand (bound by RecD)
      - RecC
        
        Recognizes chi sequence (5'-GCTGGTGG'3') which signals the halting of 3'-ending strand degradation
      - RecD
        
        Helicase motor moving 5' to 3' along one strand,
      - RuvAB
        
        Results in rapid branch migration
        
        Recruits RuvC (Holliday intermediate resolvase)
        
        RuvB= donuts
    - - Spo11
        
        Resembles type 2 topoisomerases. Active-site Tyr residue as nuc in transesterification reaction to create double stranded break.
        
        Removed by
        
        Mre11, Rad50, Xrs2
      - RPA
        
        eukaryotic ssDNA-binding protein
      - Rad51
        
        Homologous to RecA
    - - Occurs when no homologous chromosomes are present
        
        Enzymes
        
        Ku70 and Ku80 bind to DNA ends
        
        Creates Artemis complex
        
        5' to 3' exonuclease to widen double stranded break
        
        DNA-PKcs autophosphorylates
        
        Small gaps after Artemis filled by pol mu or lambda
        
        Complex XRCC4, XLF, and ligase 4 seals nicks
  - - - Fork regression occurs, resulting in 4-branched holiday junction. Lesion is paired with undamaged comp. strand and repair can occur. Holiday intermediate resolved by degradation of the short DNA arm. If lesion not repaired, the arm replicates with comp. newly synthesized strand and branch migration allows short arm to cont. replication.
- - - - tRNA
        
        RNaseP (ribonucleoprotein) removes nucleotides from 5'-end of tRNAs; the 3'-end of tRNAs is processed by one or more nucleases (ex. RNase D)
        
        CCA-3' is added after initial transcription via tRNA nucleotidyl transferase
        
        Amino acid arm: Amino acid attaches to 3' terminal A at CCA-3' terminus
        
        D arm: contains dihydrouridine
        
        TPsuedoC arm: contains ribothymidine and psuedouridine
        
        Anticodon arm: has antocodon
        
        Suppressor tRNAs allow translation to continue past nonsense mutations
      - microRNAs (miRNAs)
        
        regulate the expression of genes
        
        capped and polyadenylated after pol 2 synthesis
        
        can fold and create hairpin structures recognized by DGCR8
        
        DGCR8 recruits nuclear pri-miRNAs to Drosha (an endonuclease to cleave pri-miRNA to pre-miRNA) = microprocessor complex formation
        
        pre-miRNAs (imperfect hairpins with 1 or more unpaired bases) combine with RNA export proteins for transport out the nucleus
        
        Cleavage of pre-miRNA by dicer to release mature miRNA
        
        Dicer can also cleave long dsDNA to produce siRNAs
        
        Dicer loads miRNA and siRNA into RISCs
      - rRNA
        
        processing guided by small nucleolar ribonucleoproteins (snoRNPs) that base pair with the pre-rRNA at specific sites
        
        snoRNPs specify which methyl groups are added to 2'-OH rRNAs
      - short interfering RNAs (siRNAs)
        
        can base pair with mRNA with comp. 3'UTRs to induce degradation or translational silencing of the mRNA
    - - mRNA
  - - - Termination
        
        Termination sequences in bacteria
        
        (Rho independent) Relies on structures that form in the RNA transcript
        
        Region produces RNA transcript w/ self-complimentary sequences, creating a hairpin structure
        
        Highly conserved segment of AAA residue in the template strand near the 3' of the hairpin
        
        Hairpin formation disrupts the DNA-RNA hybrid leading to dissociation of the transcript
        
        Relies on rho (p)
        
        Includes a CA rich sequence (rut). The p factor (helicase) catches up to the rut site and contributes to the release of the RNA transcript
        
        Eukaryotic RNA pol termination
        
        Pol 3
        
        T-rich sequences in DNA toward 3' end of mature RNA
        
        Pol 2
        
        Polyadenylation sequence (AAUAA-3')
        
        Allosteric/antiterminator model
        
        Like hairpin model of bacterial termination (rho independent); conformational change of pol 2 causes it to fall off the DNA template
        
        Torpedo model
        
        mRNA extended at 3' end, and protein complexes cleave the mRNA at the polyadenylation site creating a new 3' end
        
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        Similar to p dependent
        
        pre-mRNA processing (ch 16)
        
        Capping
        
        prevents degradation by exoribonucleases at 5'-end by preventing recognition
        
        cap is a residue of 7-methylguanosine liked to 5'-terminal residue of mRNA via 5-triphosphate linkage
        
        linkage requires condensation of triphosphate at 5' end with GTP
        
        capping enzyme: guanylyltransferase
        
        plays role in binding mRNA to ribosome
        
        PolyA tail
        
        80-250 A residues on 3'-end of mRNA
        
        binding site for 1+ proteins that protect mRNA from exoribonucleases at 3' end
        
        mRNA transcript is cleaved at the polyA addition site via an endonuclease to generate a free 3'-OH group that A residues bind to
        
        splicing
        
        excision of introns based on breaking/joining of specific phosphodiester bonds
        
        spliceosome
        
        complexes of non protein-coding RNAs and proteins (ribonucleoproteins)
        
        5 small nuclear ribonucleoproteins (snRNPs) with a small nuclear RNA (snRNA) at each snRNP core
        
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        U1 snRNP binds to GU sequence at 5'-splice site (with accessory proteins U2AF) which binds to AG and flanking sequences at the 3' splice site.
        
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        some mitochondrial, chloroplast, and bacteriophage introns are self splicing
        
        Alternative splicing: creating multiple proteins from different combinations of exons
        
        2'-OH of the branch-point A residue attacks phosphate at the 5'-splice site, then the 3'-OH of the released exon attacks the 3'-splice site.
        
        Group 1 introns
        
        3'-OH of GMP/GDP/GTP attacks phosphate of 5' splice site; freed 3'-OH of 5' exon attacks phosphate of 3'-splice site
        
        group 2 introns
        
        forms lariat
      - Kinetic Proofreading (pyrophosphorolysis): ensures accuracy of transcription; catalytic rxn runs in reverse when polymerase stalls
      - Nucleolytic proofreading: polymerase reverses by 1 or a few nucleotides and breaks the phosphodiester bond upstream from a mismatched base
  - - - tRNAs charged via addition of amino acid by forming an acyl linkage b/w carboxyl group of aa and free 2' or 3'-OH of tRNA
      - Adenylation
        
        aa activated before charging with tRNA when carboxyl group of AA reacts with the alpha-P group of ATP with displacement of pyrophosphate
        
        Class 1 synthase links to 2'-OH then migrates to 3'-OH via transesterification
        
        Class 2 synthase link to 3'-OH
    - - degenerate but not ambiguous
      - Wobble base (3rd pos)allow for flexibility in genetic code; Inosine can pair with A, U, and C
      - second position normally encodes properties
      - withstands many mutations
    - - Initiation:IF-1 blocks A site, IF-3 prevents premature binding of large ribosomal subunit, and mRNA binds to the 30s subunit via its shine-Dalgarno sequence with its 5'-AUG-3' in the P site. fMet-tRNA binds to the P site with the help of IF-2-GTP. The IFs dissociate and the 50s subunit binds.
    - - Initiation: eIF3 and eIF1A (= IF-1) bind to prevent premature binding of the large subunit, and eIF1 binds to block the E site. eIF2-GTP bound to Met-tRNA (i), eIF5 and eIF5b bind to the ribosome. eIF4F has eIF4E, which binds to the 5'-cap of mRNA, and eIF4G, which binds eIF4F and eIF3 together. mRNA circulates during scanning by the 43S complex to find the start codon. Location of 5'-AUG-3' makes the 43s complex join the large subunit, and dissociation of other IFs occur.
- - - - UBF binds both, then SLI (selectivity factor 1) binds both UBFs to stabilize the complex
- - - - Consensus sequence: 5'-TATAAT-3' at -10 region and 5'-TTGACA'3' at -35 region
        
        Effects initiation
    - - 70- general housekeeping
      - 38- starvation
      - 28 flagellar structure and movement
      - 32 heat shock
      - 24 extra cytoplasmic stress
      - 54 nitrogen uptake
        
        binds to -12 and -24 regions