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BCHM218 > Module 4 Sections 1-3 > Flashcards

Module 4 Sections 1-3 Flashcards

Transcription, mRNA Processing, Genetic Code

1
Q

list all tRNAs, rRNAs, snRNAs, snoRNAs

A

snRNAs = small nuclear RNAs, play a role in gene regulation - form part of the spliceosome that is important for mRNA processing

mRNA = only type of coding RNA – determines the amino acid sequence of a protein

rRNA = ribosomal, present during translation

tRNA = transfer, present during translation

snoRNA = small nuclear RNA, involved in the processing of rRNAs

miRNA – micro RNAs, limit translation by binding to 3’-end of target mRNAs

IncRNA = long non-coding RNAs, can be important regulatory RNAs

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2
Q

transcription vs DNA replication characteristics

A

uses template strand: both

has initiation, elongation and termination: both

proceeds in the 5’-3’ direction: both

requires primers for initiation: DNA replication

has specific start and stop sites: both

is selective: transcription

rNTP’s are used as building blocks: transcription

both strands are used simultaneously as a template: DNA replication

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3
Q

the template strand

A

RNA polymerase uses 3’-5’ template DNA strand to make RNA - RNA will be complementary to the template strand but MATCH the sequence of the coding strand, replacing T with U

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4
Q

transcriptional start site

A

Where RNA polymerase starts reading

Distinct from translational start site

Which encodes the first amino acid of the protein

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5
Q

translational start site

A

Generally AUG (met)

Located many nucleotides upstream the initiation codon

Region between TSS and start codon = the 5’ untranslated region (5’UTR)

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6
Q

bacterial RNA polymerase holoenzyme subunits

A

5 polypeptide subunit core with 3 subunits (B, B’, w) and 2 alpha subunits

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7
Q

sigma factor

A

acts as a transcriptional initiating factor and adds DNA binding selectivity

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8
Q

main mechanic of RNA synthesis

A

3 aspartic acid residues in the RNA pol active site, which capture and coordinate two Mg2+ ions

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9
Q

Mg2+ ions role in RNA synthesis

A

1 of them interacts with the phosphate groups of the NTP and the other acts to bring the 3’OH of the last added nucleotide in close enough proximity to the incoming rNTP of a nucleophilic attack reaction to take place on the alpha phosphate, releasing PPi

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9
Q

does RNA polyermase have a built-in proofreading center

A

no

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10
Q

RNA polymerase alternative proof-reading mechanisms

A
  1. kinetic proofreading
  2. nucleolytic proofreading
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11
Q

kinetic proofreading

A

If an incorrect nucleotide is added, the proper H-bonding isn’t formed, which causes fraying at the DNA-RNA duplex

When recognized by the RNA pol, it can stall until pyrophosphorolysis reverses the reaction at this base pair, allowing for the correct rNTP to be paired

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12
Q

nucleolytic proofreading

A

RNA polymerase must backtrack to the mismatched base to fix the error

Must reverse its direction by a few nucleotides in the template and uses its intrinsic endonuclease activity to hydrolyze the phosphodiester backbone of the transcript upstream of the error, removing the incorrect base

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13
Q

kinetic vs nucleolytic proofreading

A

kinetic = the polymerase stalls, allowing pyrophosphorolysis to remove the incorrect base

nucleolytic = the polymerase backtracks and hydrolyzes the phosphodiester bond upstream of the incorrect base

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14
Q

phases of transcription

A
  1. initiation
    Occurs as RNA polymerase binds to specific DNA sequences called promoters

These sequences contain specific elements and are located upstream of the TSS

  1. elongation
    the process of adding nucleotides to the growing RNA strand
  2. termination
    the release oft he product RNA when the polymerase reaches the end of a gene or other transcription unit
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15
Q

5 main steps in the process of transcription

A
  1. The polymerase binds the promoter, assisted by sigma factors in prokaryotes or transcription factors in eukaryotes
  2. Open complex is formed, in which bound DNA is partially unwound near a region of 10bp upstream of (ahead of) the transcription start site
  3. Transcription is initiated within the complex, leading to a conformational change that converts the complex to the form required for elongation
  4. Promoter clearance, involving movement of the transcription complex down the DNA template and away from the promoter, leads to the formation of a tightly bound elongation complex
  5. Once elongation begins, RNA polymerase becomes a highly efficient enzyme, completing synthesis of the transcript before dissociating from the DNA template, and then recycling for a new round of transcription
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16
Q

sigma factors in transcriptional initiation

A

a transient subunit of the bacterial RNA polymerase that directs the enzyme to the promoter - different sigma factors are specific for different promoters

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17
Q

most common sigma factor

A

O^70, 70kDa as molecular weight

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18
Q

consensus sequence

A

a sequence of nucleotides or amino acids that has a similar structure and function in different organisms

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19
Q

-35 and -10 regions

A

important interaction sites for O^70
-10 region: 5’-TATAAT-3’
-35 region: 5’-TTGACA-3’

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20
Q

upstream promoter

A

AT-rich recognition element between -40 and -60 in the promoters of highly expressed genes bound by one of the subunits of RNA polymerase

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21
Q

importance of consensus sequences, UP sequences, and spacer

A

The efficiency with which RNA polymerase binds to a promoter and initiates transcription is determined by the –10, -35 and UP sequences (and the spacer), and the distance of the UP element from the transcription start site

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22
Q

spacer length importance

A

any changes to the spacer length (larger or smaller) will reduce transcription

2 turns of a double helix allows the orientation of consensus sites to be on the same side of the helix

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23
Q

increase in spacer length

A

longer than 2 turns of a double helix = consensus sites are no longer on the same side of the helix = transcription is reduced

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24
Q

steps in bacterial transcription

A
  1. initiate bacterial transcription
    - Sigma factor must bind to the promoter region, contacting the –35 and –10 sites
    - The enzymes is in the closed conformation
    - N-terminus of the sigma factor is blocking the DNA entry channel
  2. open conformation
    - Pincers of the Pol enzyme closes around DNA, and the N-terminus moving from the active site cleft
    - Independent of ATP for all sigma factors except O&54, which is ATP-dependent
  3. abortive initiation
    - RNA polymerase does not require a primer
    - Must bind and hold 2 nucleotides in place for long enough to catalyze the phosphodiester bond
    - After this bond and for the first 8-10 bonds, there is a high chance that the polymerase will release the transcript without extending it further
    - Energetically favourable for the RNA polymerase to release the 8-10 base pair RNA transcript from the initiation complex
    - Holoenzyme will begin NA synthesis again on the same template
  4. elongation
    - RNA polymerase can hold onto the transcript long enough to extend it beyond 10 nucleotides
    - At this point, the RNA becomes stable and clears the promoter to enter the elongation phase
    - RNA polymerase will continue along the DNA template until it reaches a termination signal
    - Elongation allows for the promoter sequence to clear from the polymerase
    - Weakens the hold of the sigma factor, which falls off the polymerase
  5. termination
    - Enzyme releases the DNA template only when it encounters a termination sequences
    - Polymerase is processive, moving smoothly along the template, synthesizing the complementary RNA strand and dissociating only when the transcript is complete
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25
Q

open vs closed conformation

A

closed: bound DNA is intact
open: bound DNA is partially unwound upstream of the transcription start site

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26
Q

RNA polyermases in eukaryotic cells

A
  1. RNA Pol I
    - Ribosomal RNA (rRNA) precursors
    - Responsible for transcribing rRNAs
    - 80% of all transcription
  2. RNA Pol II
    - Messenger RNA (protein coding genes)
    - Responsible for the synthesis of mRNA
  3. RNA Pol III
    - Smaller functional RNAs (tRNAs, snRNAs)
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27
Q

how many subunits does RNA Pol have for eukaryotes

A

12

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28
Q

other uses for transcription inhibitors

A

some species rely on transcription inhibitors for natural biodefense

Ex: mushroom Amanita phalloides (the death cap) produces a-amanitin, a cyclic peptide that disrupts eukaryotic mRNA synthesis by blocking Pol II

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29
Q

general structure of eukaryotic promoter

A

Core promoter that consists of a region that is recognized by the general transcription factors which are able to recruit RNA Polymerase

Has both upstream and downstream regulatory sequences

Bind gene-specific transcription factors that can act as activators (or repressors) of transcription at that locus

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30
Q

differentiation

A

the process where a cell changes from one cell type to another

31
Q

transcription factors involved in the differentiation of cells

A
  1. developing organisms starting out in a pluripotent state and move to a a higher state like a blood cell, brain cell, pancreatic cell, etc
    - they turn on specific subsets of genes to drive them towards their terminal fate (committed to a particular function, can no longer divide)
    - done by genes that regulate the transcription of other genes by binding to promoters and interacting with the transcriptional machinery
  2. using skin fibroplast to change a cell’s gene expression
    - involves induced pluripotency by pushing cell back to its embryonic stem cell state
  3. cellular reprogramming
    - using genetic engineering to force the fribroblast t oexpress the TFs or providing the fibroblasts with the actual TF protein
32
Q

pluripotent state

A

stem cells that can differentiate into cells derived from any of the 3 germ layers

33
Q

the TATA binding protein

A

binds to TATA box near position -30 of the promoter region

34
Q

TATA box sequence

A

5’-TATAAAA

35
Q

purpose of TATA box/protein

A

plays a major role in transcription initiation

36
Q

how are TATA-binding proteins recruited

A

through proteins called TBP-associated factors (TAFs) that recognize other promoter sequences

37
Q

structure of the TATA binding protien

A

Saddle-shaped protein that is able to create a sharp bend in the DNA upon binding

TBP sits on the DNA double helix like a saddle, with an extended beta sheet and loop stirrups in contact with the minor groove of the TATA box sequence

38
Q

is the structure of the TATA binding protein conventional

A

No, it is unconventional - most DNA-binding proteins recognize DNA by inserting alpha helixes into the major groove

39
Q

base pairs that are favoured in recognition sequence of TBP

A

A=T base pairs are favoured in the recognition sequence

More easily distorted to allow opening of the minor groove

40
Q

Pol II promoters have variety of

A

transcription factors
1. TFIID (composed of TBP and TAFs)
2. FIIB
3. TFII proteins

41
Q

purpose of EMSA and DNA footprinting

A

to determine the presence of a transcription factor

42
Q

EMSA

A

electrophoretic mobility shift assay

43
Q

how EMSA works

A
  1. Fragments of DNA of a known sequence are incubated with the protein of interest and then analyzed on a nondenaturing polyacrylamide gel
  2. Visualized by staining with a dye or covalently attacking a radioactive phosphate group at one end
  3. Free DNA fragments migrate more quickly through the gel than DNA bound by protein
    - A shift in migration of DNA from fast to slow in the presence of protein indicates a direct binding interaction between the protein and DNA
44
Q

EMSA results

A

an increase in protein concentration = DNA bound protein grows longer, as well as there is less unbound DNA

45
Q

what gel is used for EMSA

A

nondenaturing, since the addition of SDS (gel used for SDS-PAGE) would denature the protein, and disrupt formation of the DNA-protein interaction

46
Q

DNA footprinting goal

A

to map the exact nucleotide bases in contact with the bound protein once a direct DNA-binding interaction has been established

47
Q

DNA footprinting steps

A
  1. DNA of interested is amplified and radiolabeled at one end. One of the two PCR primers must be radiolabeled on the 5’ end to provide a point of reference for visualization
  2. Cleavage occurs at sites that are not physically protected by bound proteins. Each piece of DNA is cleaved once, generating a set of fragments that represent all possible cleavage products (except DNA that is protected by the bound protein)
  3. Resulting fragments are separated by gel electrophoresis and detected by exposing the gel to film which detects radioactive emissions from the labeled DNA fragments. Any gaps (DNA protected by bound proteins) produces a “footprint” that indicates the boundaries of the protein-binding site relative to the radiolabeled end of the DNA sequence of interest
48
Q

DNA footprinting results

A

footpring is identified by analyzing the sites of nuclease cleavage in the DNA before and after adding the protein

49
Q

processing of pre-mRNA in prokaryotes

A

Can be directly transcribed, sometimes while still being synthesized

Directly translated by ribosomes

No compartmentalization (dividing into sections) of these processes

50
Q

processing of pre-mRNA in eukaryotes

A

Transcription and pre-mRNA processing takes place in the nucleus

Mature mRNA has to exit the nucleus to the cytoplasm to be translated

51
Q

structure of mature mRNA

A

5’ cap

3’ poly(A) tail

coding region - composed of the exons spliced together with no introns included

52
Q

purpose of 5’capping

A

prevents degradation from the 5’ end

53
Q

which polymerase helps with 5’capping and how

A

RNA Pol II synthesizes single-stranded ribonucleic acids that are vulnerable to degradation at either end by exoribonucleases

54
Q

what is the 5’ cap made of

A

residue of 7-methyl-guanosine, 5’-5’-triphosphate linkage, and additional methyl groups

55
Q

5’-5’-Triphosphate Linkage

A

7-Methyl-Guanosine is linked to the 5’-terminal residue of the mRNA through an unusual 5’,5’-triphosphate linkage

Generated by an enzyme called guanylyltransferase (capping enzyme) which forms the 5’ cap through condensation of a molecule of GTP

56
Q

additional groups added to 5’ capping

A

methyl groups, added at the 2’ hydroxyl of the ribose sugars at the first and second nucleotides adjacent to the cap

57
Q

steps of 3’Polyadenylation

A
  1. RNA polymerase will transcribe the poly(A) site at the terminal end of the transcript
    - The mRNA site where cleavage occurs is marked by 2 sequence elements
    - Highly conserved sequence 5’-AAUAAA located 10 to 30 nucleotides on the 5’ side (upstream) of the cleavage site
    - A less-well defined sequence rich in G and U residues located 20-40 nucleotides downstream from the cleavage site
  2. Polyadenylation factors bind the poly(A) signal upstream of the cleavage site
    - Subsequent cleavage generates the free 3’-hydroxyl group that defines the end of the mRNA
  3. A residues are immediately added to the frere 3’-OH of the reaction
    - Bound by poly(A) binding proteins (PABPs) which physically protect the 3’ mRNA from exoribonucleases
58
Q

major difference between mRNA processing in bacteria & eukaryotes

A

splicing of pre-mRNA

59
Q

do prokaryotes have introns

A

no

60
Q

alternative splicing

A

exons in the primary transcript from a single gene are spliced together in several combinations to produce different mRNAs = different polypeptides

61
Q

what does alternative splicing give

A

different polypeptides = genetic diversity

62
Q

oligo dT primers

A

using a poly(T) sequence as a primer in the reverse transcription reaction- 3’ poly(A) tail is not present on rRNA

63
Q

characteristics of the genetic code

A
  1. Degenerate
    - Several codons have the same meaning
    - 64 unique ways to combine 4 different nucleotides in a triple codon sequence, but only 20 common amino acids
    - Multiple codons encode for the same amino acid
    - 61 coding amino acids, 3 specifies the termination of translation amino acids
  2. Robust
    - Degeneracy provides DNA with the ability to absorb single-base mutations with minimal consequences to the protein sequences it encodes
  3. Universal
    - All organisms use the same genetic code with a few minor modifications
    - Once the code evolved, it resisted change
64
Q

rules of the genetic code

A
  1. It is non-overlapping
  2. It does not contain pauses
  3. The genetic code is read in triplets
  4. It is read in a linear fashion
  5. The code is resistant to mutations
65
Q

non-overlapping vs overlapping codons

A

non-overlapping: a single nucleotide substitution would change only 1 codon = 1 amino acid change

overlapping: a single nucleotide substitution would change 3 codons = protein would have 3 consecutive amino acid changes

66
Q

the genetic code does not contain pauses experiment & result

A

Brenner and Crick used mutagens known as acridines to induce mutations in the B gene of the T4 bacteriophage - Acridines produce mutations through the insertion or deletion of a single base pair into DNA

result: Most mutations completely inactivated the B gene - Suggested that not 1 but many amino acids were changed by these mutations

67
Q

frameshit mutation

A

insertion or deletion of 1 or more paired nucleotides, changing the reading frame of codons during protein synthesis

68
Q

translation is linear rule experiment

A

looked at the synthesis of hemoglobin, using extracts from rabbit reticulocytes (immature red blood cells)

69
Q

transition mutation

A

a purine is replaced by another purine or a pyrimidine is replaced by another pyrimidine

70
Q

transition mutations positions

A
  1. third position
    - rarely cause a change
  2. second position
    - determines whether it encodes a polar (purine) or hydrophobic (pyrimidine) amino acid
    - tend to conserve the chemical nature
  3. first position
    - results in an amino acid change, but chemically similar to the original
71
Q

silent mutation

A

Single-base substitutions

Non deleterious and non-harmful

Do NOT result in an amino acid replacement

72
Q

missense mutation

A

Single-base substitution

Replaces one amino acid with another

Can be deleterious or non-deleterious

73
Q

nonsense mutation

A

Far more harmful than silent mutations

Result in a termination codon

Abort protein synthesis

Results in an incomplete protein that is rarely functional

74
Q

non-stop

A

loss of a stop codon

BCHM218 (4 decks)

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