Chapter 10

- Griffith IIIS and IIR bacteria experiment

- Avery, Colin, MacLeod Isolated transforming substance and proteases and RNases did nothing
o DNase destroyed the transforming substance
- Hershey-Chase Virus and protein and DNA with
32
P and
35
S.

Chapter 12
Meselson-Stahl experiment
- Growing E. coli on pulse of radioactive
15
N and saw that after a round of replication there was
one intermediate band between
14
N and
15
N
- Growing for a second round and then less in the intermediate range and more in the
14
N range
Unreplicated Gap Left by removal of primer at end of DNA without telomerase activity

Chapter 13
RNA pol I rRNA
RNA pol II pre-mRNA, snoRNA, some miRNA, some snRNA
RNA pol III tRNA, small rRNA, some miRNA, some snRNA

Chapter 14
Splicing
- U1 -> 5 splice site
- U2 binds to the branch point in the intron.
- The U5 and U4U6 complex joins the spliceosome
o Looping of intron so 5 splice site and branch point are adjacent
- U1 and U4 dissociate from the spliceosome
o The spliceosome is now activated
- The 5 splice site is cleaved producing exon with 3 OH
- 5 end of intron folds back and 5 -> 2 phosphodiester bond through 1
st
transesterfication
reaction w/ adenine nucleotide @ branch point
o * Lariat is formed *
- 3' splice site is cleaved and then immediately ligated to the 3OH of the first exon through the
2
nd
transesterification reaction

Polyadenylation
- Cleavage and polyadenylation specificity factor (CPSF) bind to AAUAAA consensus sequence
o Upstream of the 3 cleavage site.
- Cleavage stimulation factor (CsTF), binds downstream of the cleavage site.
- Two cleavage factors (CFI and CFII) and polyadenylate polymerase (PAP) join
o Once the complex has formed, the pre-mRNA is cleaved.
o CsTF and the two cleavage factors leave the complex.
- PAP adds approximately 10 adenine nucleotides to the 3 end
o Addition of the short poly(A) tail allows for the binding of the poly(A) binding protein
(PABII) to the tail.
o PABII increases the rate of polyadenylation, which subsequently allows for more PABII
protein to bind the tail.
Functions:
Increases the stability of the mRNA molecule through the interaction of proteins at
the poly(A) tail.
Length influences the time in which the transcript remains intact and available for
translation.
Assists with the binding of the ribosome to the mRNA.

5 Methyl Cap
- Terminal phosphate of the three 5 phosphates linked is removed
- G nucleotide is attached to the 5 end using a 5 to 5 phosphate linkage
- Methyl group is attached to position 7 of the guanine base.
o Also sometimes to original 1
st
base
- Ribose sugars of adjacent nucleotides may also be methylated at the 2OH.

Functions:
- CAP binding proteins -> 5 cap
o Stimulate binding of the ribosome to the 5 cap and to the mRNA molecule.
- The 5 cap may also increase mRNA stability in the cytoplasm.
- 5 cap is needed for efficient splicing of the intron that is nearest the 5 end

Chapter 17
RNA interference
- RNA Cleavage
o RISC + siRNA (sometimes miRNA) cleave mRNA near the middle of siRNA
This is sometimes called Slicer Activity
o mRNA is further degraded
o Increase rate of mRNA degradation and decrease rate of translation

- Inhibition of Translation
o miRNA + RISC binds to mRNA inhibiting initiation and succeeding steps.
o Most efficient with more miRNA bound to mRNA

- Transcriptional Silencing
o siRNA + RITS silences transcription by altering chromatin structure
Attracts enzymes that methylate tails of histones
o miRNA bind to complementary structure in DNA and attract enzymes that methylate
DNA directly

- Slicer Independent degradation of mRNA
o miRNA binds to AU rich element in 3 UTR is degraded by RNA-silencing mechanism
Brings about degradation of mRNA in a process that requires Dicer and RISC

Chapter 18
- Mismatch Repair
o Methylation of A on GATC of the old strand
o Mismatch repair complex brings the mismatched bases close to the GATC sequence and
nicks the old stand GATC
o Exonuclease of mismatch repair complex remove nucleotide from the nick to the
micmatch
o DNA pol and Ligase replace the DNA

- Direct Repair
o Changes nucleotides back to original structures
o Photoreactivation of pyrimidine dimers in bacteria
Photolyase Uses energy captured from the sun to break covalent bonds that
link pyrimidines in a dimer

- Base-Excision Repair
o DNA glycosylase removes the specific wrong base that it recognizes
Produces AP site
o AP endonuclease cleaves the phosphodiester bond on the 5 side of the AP site
Other enzymes remove the ribose sugar
o DNA pol + ligase

- Nucleotide-Excision Repair
o Complex of enzymes scans DNA for distortions of its 3D configuration
When the distortion is detected additional enzymes separate the 2 strands @
damaged region
o SSBP stabilize the strands
o Sugar-phosphate backbone of the damaged DNA strand is cleaved on both sides of the
damage
o Part of the DNA is peeled away by helicase enzymes and DNA pol + ligase fill the gap