C34:DNA Replication
There are multiple types of Polymerases for E.Coli, 4 of them are tasked with repair and one is tasked with replication (extending polymers). Which numbers are they?
Pol 1,2,4,5 are DNA Repair
Pol 3 is Replication
Pol 1 also does primer removal
What are Pyrimidine Dimers and how are they formed?
Usually occurs when UV radiation damages DNA. This is when neighboring pyrimidines on the same strand are linked in cyclobutane rings, the bases are joined together but cannot fit in the Helix, so it blocks translation and transcription leading to potential mutations.
Messenger RNA (mRNA): encodes proteins
Ribosomal RNA (rRNA) : most abundant
Transfer RNA (tRNA) : critical in translation
Where does transcription occur?
Inside the Nucleus
What are the key process in producing 45S pre-RNA?
Nucleotide Modification
Association with Proteins
Cleavage by RNAase 3 into smaller units
Relaxes DNA by passing intact strand through cleaved strand, reduces DNA underfunding, and DOES NOT require ATP hydrolysis to do its job.
What are the 3 basic steps involved in most DNA repair processes?
Recognition
Removal
Repair
What is the lac operon and what are the Cis-acting elements and the trans-acting elements?
Encodes proteins for using lactose as a carbon source, Lac also has groups of genes that turn genes off and on.
Cis: Promoter, Operator, and CAP protein
Trans: RNAP, Lac Repressor, and CAP Protein
Where is the TATA box commonly located and what is it's job?
Location: -25 position (in the promoter region)
Job: Is the initial binding site for TFIID, makes transcription more efficient, influences the precise spot for the transcription start site.
What Enzymes catalyze the changes that are made to the 5' and 3' ends of the precursor RNA for tRNA specifically?
5' end is removed by RNAse P, 3' end is is trimmed by RNAase Z, CCA is added to the 3' end by tRNA nucleotidyltransferase.
It goes from 3' to 5'. If an incorrect base is added, the polymerase stalls and the incorrect hydrogen bonding encourages movement, the base is removed on entering exonuclease active site, this is found in DNA Pol 1 and 3.
What is the Ames test and how is it able to identify these compounds?
They used a tester strain of salmonella that requires histidine for growth, the culture that did not contain histidine if the genome was unchanged the bacteria died. The media with the possible mutagen and minimal histidine had a high number of growth which suggests that the mutagen causes mutation.
Protein-Dependent Termination: ___ is a hexameric protein that binds to growing ___, it hydrolyzes ___ to go along the ___ until it catches the polymerase and then crashes into it, preventing transcription.
Rho, RNA, ATP, RNA
What sequence blocks does the CTD (carboxy-terminal domain) have repeats of?
1. [YTBTLS]n
2. [YSPTSPS]n
3. [LOMESN]n
4. [YRSYNLP]n
2. [YSPTSPS]n
Explain Polyadenylation. When is a tail added and what enzyme adds it?
The tail is added after release of the mRNA by Pol 2, the Poly(A) polymerase adds adenylate to the cleavage product, addition of the tail increases translation and stabilizes mRNA.
Explain the Machinery used at the replication fork and what each piece does.
Nucleases: DNA Pol 1 and 3 doing some editing
Ligase: Joining the fragments together on lag strand
Clamp Loader.
What causes double strand breaks and How are double strand breaks repaired?
Repair: 5' exonuclease generates ssDNA, there's a D-loop formation, DNA synthesis, second strand invasion AKA Holliday junction, cleavage of the junction and re-ligation.
What is Sigma's role in Initiation?
Recognizes the the promoter and guides the RNA polymerase to it, they bind and creates an open complex where DNA strands separate to leave space for access to the template strand. It won't just take any DNA they are super selective and will reduce the affinity for generic DNA.
What does the Methylation of cytosine do?
interferes with the transcription factor bind -> decreasing transcription
Recruits HDACs {deacetylated histones} -> decreasing accessibility for transcription machinery
What roles do U1,U2,U4,U5, and U6 have in splicing?
they are snRNPs, U1: base pairs with the 5' junction
U2: base pairs with the branch site
U4,U5,U6: come preassembled together and trigger a reorganization
U5: keeps exons close to one another
U6: removes U1, and binds with U2 to form the catalytic center of the complex.
What is telomerase and why is it required for linear DNA? What is the role of RNA component in telomerase?
Telomerase adds caps to the ends of chromosomes to prevent shortening and degradation of the chromosome every time it is replicated, it contains a small RNA template to provide space for the final Okazaki fragment to be placed.
Name the four DNA pathways we are required to know, and get 100 bonus points for each one you correctly explain the ingredients for (What's repaired and how).
Mismatch Repair:Misincorporation, Methylcytosine Deamination, Endonuclease, Exonuclease
Base Excision Repair:Uracil, alkylated bases, oxidized bases, Glycosylase, AP endonuclease, phosphodiesterase
Nucleotide Excision Repair: Structural distortions (pyrimidine dimers), replication slippage, Excinuclease
Direct Repair: Pyrimidine Dimers, Photolyase
Explain the key differences between intrinsic and extrinsic termination.
Intrinsic: There's a Stem-loop, that clogs RNAP leading to destabilization, downstream of the loop there is a U sequence which leads to weak base pairing.
Ultimately: U-Sequence + Stem Loop = RNAP Dissociation
Extrinsic: Rho uses ATP hydrolysis to advance and catch the RNAP and splits the RNA-DNA hybrid.
Explain the Roles of Enhancers, Coactivators, and Mediators
enhancers: regulate transcription over long distances, bound by transcription factors and are tissue specific.
coactivataors: recruited after transcription factors bind, interact with other proteins in large complexes to facilitate transcription, modulators.
Mediators: a huge bridge between transcription factors and Pol 2, has tons of subunits, and can promote the phosphorylation of Ser 5 and the CTD.
How does self-splicing occur for RNA?
bond is broken within the intron, and the intron becomes attached to a free guanosine nucleotide or an adenosine nucleotide within the intron itself, then the two exons are joined together releasing the intron.