Lecture 4 - 6 (Ch 8) Flashcards
The problem of linear DNA replication is ___
stranded RNA primer at the end of the lagging strand after RNA primer removal
Without telomere activity, this overhang will be ___, leading to ___
removed, leading to the lagging ends of the daughter strand to be shorter than the parent
Telomeres are ___; they are synthesized by
repetitive DNA sequence end of chromosome, synthesized by telomerase
With the telomeres, the chromosome (is/isn’t) shortened; how?
is; lack of telomerase means the telomeres won’t be remade! However, it will not effect vital genome
Telomerase creates telomeres in 4 steps:
- Telomerase (containing repetitive RNA sequence) attach to the 3’ overhang (longer parent sequence) 2. Telomerase RNA acts as the template for DNA elongation of the 3’ overhang (in the 5’->3’ direction) 3. Telomere translocates down the overhang 4. Step 2 repeats, causing DNA elongation 5. DNA polymerase fills the resulting “gap” in the daughter sequence*
Telomerase RNA sequence runs in ___ direction
3’->5’
3 Steps of PCR are:
- Denaturation of dsDNA to ssDNA via heat 2. RNA Primer annealing at a lower T 3. Primer extension via Taq DNA Polymerase and dNTP
PCR is carried out in (one/both) DNA strands
both
In PCR, helicase, SSB, primase, ligase, and sliding clamp is not needed because:
Helicase and SSB: heat creates ssDNA, so neither are needed Primase: RNA primer of interest provided manually Ligase: No Okazaki fragments created, because ssDNA rather than dsDNA opening up Sliding Clamp: Taq has ++ efficiency, and sliding clamp is not heat stable
Deoxynucleotide DNA Sequencing: DNA v. dDNA?
DNA has 5-carbon ring with 5’-Phosphate and 3’-OH dDNA has 5-carbon ring with 5’-Phosphate and 3’-H Therefore dDNA cannot form P-d bonds
ddNTP DNA Sequencing: 3 key ingredients & their functions are ___
- 5’ primer labeled with radioactive 32P (for later visualization on gel) 2. ddNTP (one base per condition) 3. dNTP (all 4 bases in all conditions)
ddNTP DNA Sequencing: DNA fragment stops are by ___
chance
ddNTP DNA Sequencing: After performing all 4 conditions, describe the gel and its results. The sequenced strand is the ____ strand. To read the original strand of interest, ____
4 lanes for 4 bases of ddNTP. Furthest is the smallest (closest to 5’ primer). Read the sequenced strand bottom-up, creating 5’->3’ complement; complement; original strand is 3’->5’ complement of the sequenced strand
Next Gen DNA Sequencing has 3 characteristics:
- smaller DNA sample 2. sequence by synthesis - each time a new nucloetide is added, the whole seunce stops; the base is recorded via flourescence, and ddNTP exchanged with dTP 3. Sequence of many targets
mRNA in ____ were located through the pulse-chase experiment, in which ___
Eukaryotes; radiactivity of uracil was tracked from the nucleus to the cytosol
Commanalities between DNA and RNA synthesis (3):
5’->3’; dependent on DNA template; P-P elimated with each P-d bond
Conservation between bacterial and euakryote transcription is ____, but not its ___.
RNA polymerase structure, but not its units
RNA polymerase structure that is analog in bacteria and in eukaryote are: ___; non-conserved structures are: ___
5 subunits (alpha I, alpha II, beta, beta’, omega); sigma
In bacteria, the sigma subunit ___; in eukarya, the sigma subunit ____.
changes for the type of promotor it binds to; does not exist, but other additional subunits exist
In bacteria, the subunit that gives RNA Polymerase specificity is the ___
sigma subunit
In eukarya, the subunit that gives RNA Polymerase specificity ___
does not exist; rather, there are multiple RNA polymerases for different functions.
The two differences between RNA Polymerase in bacteria and in eukarya are:
- one RNA polymerase modified by sigma subunit in bacteria, and multiple RNA polymerase in eukarya (to recognize different promotors) 2. Eukarya requires RNA complex
Eukarya have (less/more) diverse promotor sequences and consensus sequences that bacteria
more
Eukarya: 3 RNA Polymerases are:
- RNA Pol I 2. RNA Pol II 3. RNA Pol III
Eukarya: Explain the functions of 3 RNA Polymerases:
- RNA Pol I: transcribes 3 rRNA genes 2. RNA Pol II: transcribes protein coding genes and most snRNA (used in RNA processing) 3. RNA Pol III: transcribes tRNA, 1 snRNA, and 1 rRNA
Promoter are identified by ___
ID’ing protein-coding genes and comparing shared DNA; for example, the TATA box
TATA box are promoters with the sequence ____, ___ nucleotides away from +1 position
5’ TATAAA 3’; -25
Consensus sequences were proven to have function in an experiment of 3 conditions. What were the 3 conditions? What was the indicator that the promotor had a function?
inserting cloned plasmid 1. without promotor; 2. with a promotor; 3. with a point mutation promotor into the cell, and looking for colored product of LacZ
In Myers LacZ experiment, was the unmutated promotor found to be necessary and sufficient for gene expression?
No– point mutation still indicated LacZ activity, but in smaller amounts
If an ori was introduced to a plasmid, the plasmid will ___
replicate
In Myers LacZ experiment, promotor was added to ___ w/n the plasmid; this is because it contains many ___
Multiple Cloning Sites; restriction enzyme sites
In Myers LacZ experiment, point mutation was mapped out, and consensus sequences were elucidated– how?
consensus sequences were visualized as clusters of nucleotides that lowered expression when point mutation was introduced
The relationship between RNA Pol II, Transcription Factor II, and promotor
RNA Pol II recognizes and binds to promoter sequence with the aid of proteins called TFII (the II refers to RNA Pol II)
____ is the principle binding site during promotor recognition, which is highly conserved in many/most protein encoding genes
TATA
In Myers LacZ experiment, certain parts of the gene was protected from mutation by ___. Success of this assay was done through (1) ___, which separated DNA by ___. An alternative confirmation can be done by (2) ___, which ___
A protein covering it; (1) Gel shift assay; weight (protein-DNA > DNA) (2) Chip assay, which adds antibody to known protein
What are the four steps to RNA Pol II attaching to the promotor?
- TFIID (TAF+TBP) bind to TATA box 2. Minimal initiation complex forms with the addition of TFIIA, TFIIB, RNA Pol II, and TFIIF 3. Pre-initiation complex forms with the addition of TFIIE and TFIIH 4. RNA Pol II is released from the GTF’s to begin transcription
TFIID contains two things: ___
TBF (TATA-box Binding Protein) & TAF (TBF-Associated Factor)
The minimal initiation complex contains five things: ___. Out of these, only ___ was present on the DNA from the previous step
TFIIA, TFIIB, TFIID, TFIIF, and RNA Pol II; TFIID
