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BIO230 > Lecture 2 > Flashcards

Lecture 2 Flashcards

1
Q

what are genomes? what has genomes? what are genomes made of?what are the genomes for viruses? what is genome expression required for?

A
  • Encodes the information to construct and maintain an organism
  • All known life forms possess a genome
  • Most genomes are made of DNA
    ➢ viruses aren’t living (don’t have cells but infect cells)
    ➢ except some viruses have RNA genomes where some have DNA genomes
  • genome expression is required to release of the biological information stored in the genome
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2
Q

what is the transcriptome? what molecule does it read? what tool is used to read one?

A
  • The first product of genome expression is the transcriptome
  • It is the repertoire of RNA molecules present in a cell at a particular time
  • use the DNA microarray to read the RNA transcriptome
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3
Q

what is the DNA Microarray? how do you read it? what is a more advanced tool do we now use instead of DNA microarrays?

A

gives you the snapshot of RNA transcriptome

you read the microarray as a table. rows are the genes (A,B,C etc) and columns are samples from different parts of body.

depending on the color in the intersection you can see how much RNA is present:
red = lots of RNA
green = little RNA
black = middle amount of RNA

Note: We now use RNAseq more for transcriptome analysis rather than DNA Microarray (misleading because it measures RNA lol)

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4
Q

what maintains the transcriptome

A
  • the transcriptome is maintained by the process of transcription
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5
Q

what is the proteome?

A
  • The second product of genome expression is the proteome (first product is the transcriptome)
  • It is the collection of proteins in a cell
  • Defines the biochemical functions of the cell
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6
Q

what is 2D gel electrophoresis used for? how do you read it

A

gives you a snapshot of the proteome

read it like a graph
- y-axis is measured based on molecular weight from low to high
- x-axis is measured from acidic to basic with the isoelectric point in between
- size of the splotch represents the amount of that protein represented in the cell (may be different between samples areas even if taken from same dna)

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7
Q

what maintains the proteome?

what is central dogma?

A
  • maintained by the process of translation

central dogma:
Genome (DNA) ➔ Transcriptome (RNA) ➔ Proteome (Protein)

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8
Q

how do we produce different cell types?

A

Differences in genome expression!

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9
Q

stats about genome expression:
- how many genes in the human genome
- what percent is expressed
- variation between cells?

A
  • Human genome ~25,000 genes
  • At any one time only 30-60% of genes expressed
  • Expression of almost all genes varies from one cell type to another
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10
Q

how do you regulate genome expression? draw the map out

whole map is on slide 11

A

location: Genome:
Dna is organized –>

transcription –>

location: Transcriptome:
post transcriptional sequence-> RNA -> splicing –>

Translation –>

location: Proteome:
post translational –> protein –> localization (via sorting) –>

location: interactome –> location: metabolome

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11
Q

why is regulation of gene expression crucial

A
  • Defining Cell Types
    (multicellular organisms)
  • Responses to extracellular
    stimuli (both multicellular and
    unicellular organisms)
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12
Q

recall eukaryotic transcription – chat

A
  • Location: Occurs in the nucleus of eukaryotic cells.
  • Transcription factors: Proteins that help bind the RNA polymerase II to the DNA.
  • Promoter: Specific DNA sequence (e.g., the TATA box) where RNA polymerase binds to initiate transcription.
  • RNA polymerase II: The enzyme responsible for synthesizing mRNA (messenger RNA) from the DNA template.
  • Initiation: Transcription factors and RNA polymerase II assemble at the promoter region to begin mRNA synthesis.
  • Elongation: RNA polymerase moves along the DNA, adding RNA nucleotides complementary to the DNA template strand.
  • Termination: Transcription ends when RNA polymerase reaches a termination signal, releasing the newly synthesized mRNA.
  • mRNA processing: After transcription, the pre-mRNA undergoes:
  • 5’ capping: Addition of a modified guanine at the 5’ end.
  • Splicing: Removal of introns (non-coding regions) and joining of exons (coding regions).
  • Polyadenylation: Addition of a poly-A tail at the 3’ end for stability.
  • Final product: Mature mRNA is exported from the nucleus to the cytoplasm for translation.
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13
Q

recall prokaryotic transcription - chat

A
  • Location: Transcription occurs in the cytoplasm (no nucleus in prokaryotes).
  • DNA to mRNA: DNA is copied into mRNA (messenger RNA) by the enzyme RNA polymerase.
  • Promoter: Transcription begins when RNA polymerase binds to a specific DNA sequence called the promoter.
  • Sigma factor: A subunit called the sigma factor helps RNA polymerase recognize and bind to the promoter.
  • Initiation: Once bound, RNA polymerase unwinds a short section of DNA.
  • Elongation: RNA polymerase moves along the DNA, synthesizing a complementary RNA strand by adding ribonucleotides.
  • Termination: Transcription ends when RNA polymerase reaches a terminator sequence, causing the RNA strand to be released.
  • Polycistronic mRNA: In prokaryotes, a single mRNA can encode multiple proteins, a feature known as being polycistronic.
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14
Q

what does it mean by genes can be transcribed at different efficiencies? what is another term for this and what is used to do this?

A

Gene expression in both prokaryotes and eukaryotes is regulated by gene regulatory proteins (transcription factors) –> control how much RNA is made
- you could have two genes on the dna, and both will go through transcription to make RNA, but one may make much more RNA than the other.

thus more RNA translates to more of one type of protein. This difference in efficiency is due to different regulation of genes.

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15
Q

what do gene regulatory proteins (transcription factors) regulate?

where doe the proteins bind?

what are the two modes the proteins can conduct on the gene?

A

Gene expression is controlled by gene regulatory proteins (transcription factors)

gene regulatory proteins bind specifically to regulatory regions of DNA known as cis elements

  • cis elements = specific DNA sequences located near the genes they regulate - usually near promoter

Gene regulatory proteins can turn genes:
➢ ON = Positive regulators = activators
➢ OFF = Negative regulators = repressors

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16
Q

review ecoli as a bacterial gene regulation model

  • what is ecoli
  • how many proteins does it encode for
  • how are the genes regulated
  • what is the prokaryotic feature of ecoli
A

E. coli:
* unicellular prokaryote
* one chromosome of circular DNA
* encodes about 4300 proteins
* many genes are transcriptionally
regulated by food availability

Prokaryotic feature:
* Multiple genes can be transcribed into a
single RNA molecule
➢This system is called an operon

17
Q

example 1: Tryptophan (Trp) Operon (for ecoli):
- how many genes are in the operon?
- what does it encode for?
- how many promoters regulate transcription?

A
  • Five genes on the ecoli chromosome
  • Encode enzymes for tryptophan biosynthesis
  • Transcription regulated by a single promoter

as stated on the last slide, the prokaryotic feature of ecoli is that it can transcribe into a single RNA molecule via an operon which creates a series of enzymes required for tryptophan biosynthesis

18
Q

What are The Tryptophan (Trp) Operon Promoter’s two potential protein-bound states and how does it affect gene expression?

A

1) Bound by RNA polymerase
➢ Trp gene expression ON

2) Bound by a tryptophan repressor protein
➢ Trp gene expression OFF

The tryptophan repressor binds to a specific DNA sequence of the
promoter called an operator.

19
Q

How does the tryp repressor work?

how is the tryp repressor regulated?

what does the repressor and operator act like?

A

Tryptophan repressor binding blocks promoter access:
➢ RNA polymerase cannot bind
➢ Negatively regulates Trp expression

BUT, tryptophan repressor DNA-binding activity is
regulated. The repressor must bind two molecules of tryptophan to bind to DNA.

The repressor and operator provide a simple switch to control tryptophan biosynthesis according to the availability of free tryptophan

20
Q

what is the motif that is found twice on the tryptophan repressor and what does it do?

A

Tryptophan repressor contains a helix-turn-helix DNA binding motif
(most common DNA-binding motif)
- this common shape is found on the repressor and allows it to bind to the major groove on the DNA double helix’s operator region to induce conformational change and repress tryptophan creation

21
Q

summarize how the tryptophan operon works with low and high tryptophan

A

THE DNA
- 5 genes on the DNA of Ecoli
- the operator is located in between the promoter sequences
- transcription starts at +1

LOW TRYP
- the repressor is inactive and does not bind
- thus RNA polymerase can bind to the promoter region
- the operon is ON and mRNA is synthesized to make tryp enzymes

HIGH TRYP
- the 2 tryptophan molecules bind to the active repressor
- the RNA polymerase cannot bind
- the operon is OFF and mRNA is not synthesized to make tryp enzymes

22
Q

example 2: the ecoli lac operon

how many genes are required to transport lactose into the cell and what is it used for?

the lac operon enables the use of lactose in the absence of what?

what are the three conditions for lactose to be used instead?

A

E. coli Lac operon:
* three genes required for transport of lactose into the cell and for its catabolism
* enables use of lactose in the absence of glucose

when is the lac operon used:
1. Ecolis first choice is to use glucose
2. When there is low glucose, and high lactose, it will then use lactose. both those conditions must be true to use lactose
3. The lac operon is what is turned on when it wants to use lactose

  • dual regulation: both positive and negative control
23
Q

what is the activator and repressor for lac operon and what does it promote/inhibit

A

1) Activator: Catabolite Activator Protein (CAP)
Promotes Lac expression: low glucose/high lactose
2) Repressor: Lac Repressor Protein
Inhibits Lac expression: low lactose

24
Q

where does RNA polymerase bind on the lac operon ecoli stretch

A

promoter – RNA polymerase binding site

25
Q

where does lac repressor bind on the lac operon ecoli stretch

A

lac operator after the promoter region next to the transcription region

26
Q

where does CAP bind on the lac operon ecoli stretch

A

cis-reglatory sequence specifically for CAP before the promoter region

27
Q

what does the lac operon first gene encode for (what does the transcription do)

A

1st gene of Lac operon encodes β-galactosidase; breaks down lactose to glucose and galactose

28
Q

when does the lac repressor bind to the lac operator

A

when HIGH glucose, and LOW lactose –> repressor binds to the operator, the operon is OFF, and glucose is used instead

if lactose is increased the repressor will be removed from the operator

29
Q

when and how is the lac repressor removed from the lac operator

A

when:
- if lactose is increased the repressor will be removed from the operator (HIGH glucose, HIGH lactose)

how:
- when HIGH lactose, HIGH allolactose (related to lactose; requires β-galactosidase)
- allolactose binds to the lac repressor and:
➢ Conformational change
➢ Decreases DNA-binding activity
➢ Release from the operator

  • Note: operon is still OFF as glucose is still high even though lactose is high
30
Q

why is an activator needed for RNA polymerase to bind to the promoter in the lac operon. what is this activator called and what motif does it contain (note: not needed for tryp operon)

A
  • RNA polymerase binding is inefficient to the Lac promoter (lac promoter are not good binding sites and requires help)
  • Efficient RNA polymerase binding to Lac promoter requires CAP protein to be
    bound
  • CAP protein contains a helix-turn-helix DNA binding domain
31
Q

when is the CAP activator stimulated/activated? How is the CAP activator bound to the CAP binding site?

thus when
Glucose dec = ? inc
Glucose inc = ? dec

A
  • CAP DNA-binding activity is activated by LOW glucose - How?
  • Decreasing glucose levels increase the levels of a signaling molecule
    called cyclic AMP (cAMP)
  • cAMP binds to CAP protein:
    ➢ Conformation Change
    ➢ Increases DNA-binding activity
    ➢ CAP binds to CAP-binding site
  • CAP recruits RNA polymerase to bind to the Lac promoter

Thus
when:
Glucose dec = cAMP inc
Glucose inc = cAMP dec

32
Q

summarize how the lac operon turns on

A

conditions state that LOW glucose, HIGH lactose –> operon is ON

lactose is HIGH = allolactose is HIGH:
- allolactose binds to the repressor = releases it from the operator
- the repressor cannot bind DNA
- RNA pol. can bind but needs to help…:

glucose LOW = cAMP HIGH:
- cAMP binds CAP protein
- CAP protein binds to the DNA on the cis-regulatory sequences for CAP
- RNA pol. can bind to promoter sites

THUS, operon ON and transcription is on to transport lactose in the cell

BIO230 (15 decks)

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