DNA replication

Termination: Rho-independent: Rho-dependent:

1) Dna template has a self-complementary sequence (rich in CA bases) on which Rna hairpin formation occurs.

2) Rho helicase binds to rut sequence and migrates 5' to 3' along the Dna/Rna duplex until it encounters the transcription complex (paused at termination site).

3) Rho's helicase activity contributes to Rna release and promotes translocation along Rna (hydrolysing ATP).

RNA POLYMERASE

In eukaryotes there are 3 different Rna polymerases (I: for pre-rRna, II: for mRna, III: for tRna and 5S rRna) each has a specific function and requires a specific promoter.

Rna pol II (12 subunit enzyme with a carboxyl-terminal domain (CTD) tail) requires many other proteins to perform transcription + recognises several regulatory sequences.

Transcription factors regulate Rna pol's affinity for promoter & access to genes in order to control.

1. Transcription rate.
2. Activating & repressing proteins ex: histone modifications (by histone acetylase & deacetylase)

POST-TRANSCRIPTIONAL MODIFICATIONS

These modifications are necessary to protect mRna from ribonucleases, for the binding of specific proteins regulating transcription, for the coordination of transcription and translation.

5’-CAPPING: operated by enzymes associated to the CTD of Rna pol. Occurs early on in transcription protects from ribonucleases and binds to a specific cap-binding complex of proteins and participates to the binding of mRna to the ribosome (so as to initiate translation)

3’-POLY(A) TAIL: transcription extended beyond site where tail is added cleavage by endonuclease component of enzyme complex associated to CTD tail of Rna pol II, at a highly 5’- 3’ site (upstream of cleavage point), conserved AAUAAA- and a less well-defined sequence cleavage generates a free 3’-OH rich in G & U (downstream of

(cleavage point) A residues (doesn't require a template, just a cleaved mRNA as a primer) added by a poly(A) polymerase

The poly(A) tail may help protect mRNA from enzymatic destruction & help coordinate transcription and translation. →SPLICING: removal of introns and joining of exons temporal separation between transcription and translation + increased gene flexibility can generate miRtrons with regulatory functions & don't require ATP,

There are 4 classes of introns: group I and group II introns are self-splicing, group IV introns require ATP & endonucleases and ligases, other group introns require ATP & spliceosome.

Introns are removed one at a time in 5’ → 3’ direction (while the transcript is still associated with RNA pol.); the chemical reactions that take place are transesterifications.

→Spliceosome: complex made up of snRNPs each contains a class of snRNAs (U1, U2, U4, U5, U6) introns have GU at 5’ end and AG at 3’ end

that are removed by the spliceosome usually

DNA REPAIR

I has an exonuclease site (in 3’→ 5’ direction).

1. PROOFREADING: (during replication) Rna pol →If an incorrect nucleotide is added it stops and positions it in the site removes it and realignsinsertion sites as to allow for a new nucleotide to be placed.
2. ON THE NEO-SYNTHESISED STRAND:
   • • →MISMATCH REPAIR: Muts + MutL assemble at mismatch site (ATP use) MutHbinds and Dna is threaded through complex until a hemimethylated GATC sequence→is encountered MutH cleaves G of GATC sequencecleavage (5’ side):Mismatch is upstream of Mismatch is downstream→ of cleavage (3’ side): DnaDna helicase II + exonuclease VII or RecJ→Degradation of segment Dna pol II + SSB helicase II + exonuclease I→ →Ligase or VII or X degradation of→Sequence Dna pol III + SSB→ Ligase
   • • BASE-EXCISION REPAIR: Dna glycosilase breaks glycosidic bond between base→ →and sugar AP site

(abasic site) AP endonuclease removes sugar and adjacent→nucleotides

Dna pol I replaces nucleotides and the ligase seals nick•

NUCLEOTIDE-EXCISION REPAIR: (for larger distortions in the helix)bps + 5 nucleotides at 3’ end + 8 nucleotides at 5’ end →[ in E. Coli] 1/2 aberrant12/13 bp sequence removed.

→1/2 aberrant bps + 6 nucleotides at 3’ end + 22 nucleotides at 5’ end[ in humans]28/29 bp sequence removed.

→[E. Coli] ABC EXCINUCLEASE (UvrA, uvrB, UvrC): A B assembles at lesion site A2 2→ UvrB makes excision at 3’ end & UvrCdisassembles & UvrC associates with UvrB (remaining)→ →at 5’ end UvrD helicase removes sequence Dna pol I & ligase.