Brainscape's Knowledge GenomeTM

Browse over 1 million classes created by top students, professors, publishers, and experts.


See full index

BCHM 218 Part 2 > Module 4 Section 1 > Flashcards

Module 4 Section 1 Flashcards

1
Q

DNA replication and transcription similarities

A
  1. Uses DNA as template
  2. initiation, termination, elongation steps
  3. 5’ to 3’
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
2
Q

DNA replication and transcription differences

A
  1. rNTPs used (or just NTPs)
  2. selective (environmentally responsive)
  3. No primers
  4. Only one strand used as template
    - 5’ to 3’ coding stand (nontemplate)
    - 3’ to 5’ template strand
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
3
Q

Transcriptional and translational start sites

A
  • space between the start sites for transcription and translation (AUG) called the 5’ UTR
  • Prokaryotes: 10-30 nucleotides
  • Eukaryotes: >100 nucleotides
  • UTR typically has regulatory purpose for initiation of transcription/translation
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
4
Q

Bacterial RNA Pol.

A
  • clamp structure wraps around DNA
  • structure conserved for eukaryotic RNA Pol. I, II, III
  • Holoenzyme= 5 subunit core enzyme plus sigma factor co-enzyme subunit
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
5
Q

Mechanics of RNA synthesis

A
  • three Asp (D) residues in RNA pol active site, capture and coordinate two Mg++ ions
  • 1st Mg++ deprotonates 3’-OH
  • one Mg++ interacts with phosphate groups of rNTP and the other Mg++ acts to bring the 3’OH of the last nucleotide close enough to incoming rNTP for a nucleophilic attack on alpha-phosphate
  • this releases PPi (pyrophosphate), 2nd Mg++ facilitates departure
  • assisted by H-bonding between incoming rNTP and template DNA, allowing precise alignment of active site
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
6
Q

RNA Pol kinetic proofreading

A

-speed: 50-90 nucleotides/s
-Error rate: 1 in 10^4-10^5 (DNA pol ~10^6)
-error rate is acceptable b/c RNA is short lived
if incorrect nucleotide added:
-incorrect H-bonding, frays DNA-RNA duplex
-enzyme stalls
-pyrophosphate moves in, strips the incorrect NTP (called pyrophosphorolysis)

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
7
Q

RNA Pol nucleolytic proofreading

A
  • occurs if RNA Pol does not stall after mismatch
  • RNA-DNA strand will begin to fray @ point of mismatch
  • RNA Pol with freeze and backtrack
  • peels fraying end into rNTP entry channel
  • water hydrolyzes the phophodiester bond, which cleaves it
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
8
Q

Steps of transcription

A
  1. binding of RNA pol. core to the DNA promoter
  2. Formation of transcription bubble
  3. Initiation
  4. Elongation (promoter clearance)
  5. termination + recycling
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
9
Q

Sigma factors

A
  • RNA Pol is generic enzyme
  • sigma factors allow for selective, context-specific transcription
  • ie. sigma-70 is most common in E.coli, activates ‘housekeeping’ genes, the consensus sequence allows for sequence ‘promiscuity’ (doesn’t have to be perfect)
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
10
Q

Sigma factors: consensus sequences

A
  • changing certain base pairs in the promoter to match the consensus sequence can increase transcription
  • some differences from consensus may be better than consensus (more transcription) - called superconsensus
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
11
Q

UP sequences, -10/-35 and spacer

A

Determines efficiency RNA Pol. binds to DNA:

  • UP, -10, -35 sequences
  • the spacing between -10/-35 and the UP element
  • the distance of the UP element from the +1 site
  • spacer between -10 and -35 is 17-20 nucleotides, depending on the factor (roughly 2 turns of helix)
  • changes in spacer length with reduce transcription
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
12
Q

Transcription Initiation Steps (4)

A
  • primer independent
    1. Start at closed complex
  • holoenzyme binds a certain area of DNA but DNA strands still together
  • N-term of sigma-factor blocks the DNA entry channel
    2. Open complex (ATP independent)
  • form 17 nucleotide bubble as DNA opens
  • N-term of sigma-factor moves
  • Pol clamps around DNA
    3. must bind and hold two nucleotides in place for long enough to catalyze phosphodiester bond (unstable for first 8-10 bonds)
    4. Promoter clearance
  • Pol clears promoter
  • RNA is exiting out RNA exit channel
  • sigma-factor falls off
  • Pol. moves until termination
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
13
Q

Abortive initiation

A
  • within first 8-10 nucleotides, the RNA oligomer is highly unstable
  • if the Pol releases transcript without extending it further than the <8-10 nucleotides, called abortive initiation
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
14
Q

Different RNA pol. functions in eukaryotes and inhibitor

A

Pol I: produces rRNA
Pol II: produces mRNA, microRNA, noncodingRNA
Pol III: tRNA, some rRNA
-Pol II is inhibited by alpha-amanitin

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
15
Q

Eukaryotic transcription

A
  • more sophisticated genome, so transcriptional regulation is more sophisticated (than prokaryotes)
  • TATA box used as a promoter for some, but not all promoters (all Pol. still require the TATA binding protein (TTB))
  • Bacterial RNA Pol. and Euk RNA Pol. very similar at core
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
16
Q

Typical eukaryotic promoter layout

A
  • Binding sites can be found upstream, downstream, within genes (transcription factors=sigma factors in Euk)
  • General transcription factors bind the core promoter region
  • Transcription activators and co-activators bind regulatory sequences (more gene specific)
  • core promoter includes TATA box 25% of the time (5’-TATAAA-3’)
17
Q

TATA Binding protein

A
  • Essential to transcription of ALL genes, including those lacking TATA box in core promoter
  • binds minor groove by inserting 2 Phe residues between BP, bends DNA, opens up minor groove
  • binds to A-T b/c easier to distort
  • enables sequence-specific H-bonding
18
Q

Naming transcription factors

A
  • for general TF, it indicates the RNA Pol. that is involved
  • ie. TFII indicates transcription factor for RNA Pol. II
  • individual factors distinguished by letter afterwards
  • ie. TFIIA
19
Q

Typical layout for Eukaryotic RNA Pol. II core promoter

A
  • TATA box (-30) sometimes
  • TFIIB recognition element (BRE) 5’ of TATA box
  • Initiation sequence (INR) (+1)
  • Downstream promoter element (+30)
  • these all allow for recruitment of RNA Pol. II
20
Q

Regulatory sequences in complex eukaryotes

A
  • DNA looping allows contact between the core promoter and distant regulatory elements
  • even if thousands of BP away, packaging could have right next to each other
21
Q

Nuclear Reprogramming

A

-Pluripotent and embryonic stem cells can become any cell in body
-as cell differentiates, you get certain gene expression profile that designates it as specific cell (ie. heart, neuron)
Induced pluripotency: return differentiated cell to embryonic stem cell-like state
Direct reprogramming: Convert to another differentiated cell type by introducing different transcription factor (iPSC induced pluripotent stem cells)

22
Q

EMSA technique

A
  • fragments of DNA of known sequence are incubated with protein of interest, then analyzed on non-denaturing (protein not denatured) polyacrylamide gel
  • DNA used in experiment visualized by staining with dye or attaching radioactive phosphate group to end
  • free DNA fragments migrate through gel more quickly than protein bound DNA
23
Q

Ways to amplify only cDNA with PCR/qPCR

A
  • at least one primer spans an exon-exon boundary

- OR the PCR product spans the boundary (if introns are too long to fully replicate during the PCR cycle

24
Q

DNA Footprinting

A

-used to map the exact nucleotide bases in contact with the bound protein

25
Q

DNA footprinting steps

A
  1. DNA of interest amplified+radiolabelled at one end (one of the two PCR primers has to be radiolabelled on the 5’ end to provide point of reference)
  2. Cleavage occurs only at sides that are not physically protected by the bound protein
    - each piece of DNA cleaved just once on average, generating set of fragments that represent all possible cleavage products
  3. DNA+ protein are separated by denaturing gel elecrophoreisis, visualized by exposing gel to film that detects radioactive emission
    - gaps in cleavage sites indicates “footprint,” region where protein is bound
    - one side is DNA alone (all possible cleavage sites), other side is DNA + protein (without protein attached), has footprint on this side

BCHM 218 Part 2 (13 decks)

Brainscape helps you reach your goals faster, through stronger study habits.
© 2024 Bold Learning Solutions. Terms and Conditions