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Molecular Biology > Gene Regulation > Flashcards

Gene Regulation Flashcards

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AP® Biology flashcards Biology 101 flashcards
1
Q

Histones modification

A

• Monomethylation can be repression or expression
• Bi or trimethylation usually means repression
• Closed chromatin -> dna inaccessible -> transcription repressed
• Histone acetyltransferase causes acetylation
• Relaxes and opens chromatin
• Promotes expression
Condensation = repression
• Histones are highly basic (+ve charge) proteins
• Bind dna phosphates and prevent transcription
• Relaxation = activation
• Acetyl groups have -ve charge
• When acetylated, histones lose their charge and dna dissociates

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2
Q

Methylation:

A

• CpG islands are C and G rich regions
• In humans about 70% of promoters have cpg islands
• Different cell types have different methylation patterns (different gene expression)
• Methylation is an important component in numerous cellular processes, including embryonic development and X chromosome inactivation
A large amount of research on dna methylation and disease has focused on cancer and tumour supressor genes
• Methylated mRNA predominantly compresses of m6A (N6-methyladenosine)
• M6A modifications increase rna turnover and reduce protein levels , but there are exceptions
• M6A can also impact pre-mRNA processing and translational efficiency in specific contexts
• How RNA methylation can alter transcript fate is poorly understood

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3
Q

• RNA pol I

A

transcribes ribosomal rnas (rRNAs)

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4
Q

• RNA pol III

A

transcribes small RNAs, e.g. tRNAs

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5
Q

• RNA pol II pre-initiation complex

A

transcribes RNAs that will become mRNAs
• Pre-initiation complex:
• Regulatory elements include promoter, promoter proximal elements and enhancers (can be far from promoter)
• Mediator modulates TFIIH activity (gets it to the complex)
• TFIIH is a general TF that acts to recruit RNA pol II to the promoters of genes
• It functions as a helicase that unwinds dna

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6
Q

GAL regulation in yeast

A

• Not an operon but similar mechanism
• Catabolic pathway
• All genes under control of same TF
• GAL genes should only be expressed when galactose is present
• GAL80 is GAL4 repressor
• Galactose binds to GAL3 that gets rid of GAL80
• GAL80 : repressor that binds GAL4
• GAL3: binds galactose and GAL80
• GAL4: activator

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7
Q

• Use of GAL4-UAS system in drosophila:

A

• GAL4 lines express GAL4 in a subset of the animals tissues
• Reporter lines of fruit flies express the UAS (upstream activation sequence that gal 4 binds to) region next to the gene of interest
• In a percentage of the offspring, the desired tissue will express the gene of interest
• Can be used to knock down genes in specific tissues
• Can also make expression of your desired gene temperature sensitive by including a temperature sensitive version of the repressor GAL80

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8
Q

• Splicing:

A

• Coding sequences in dna are often interrupted by intervening sequences (introns)
• One gene can code for multiple isoforms of a protein
• Splicing occurs in nucleus
• Only shown in RNA pol II transcripts
• SnRNPs (snurps) or small nuclear ribonucleic particles are RNA-protein complexes
• Roughly 95% of multi-exotic genes undergo alternative splicing in humans
• Most common form is exon skipping

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9
Q

Regulation by micro-RNA (miRNA):

A

• MiRNAs are transcribed by RNA pol II
• MiRNA biogenesis depends on:
• PRI-miRNA transcription
• Pri-miRNA levels correlate with mature miRNA
• Processing by drosha and dimer in the nucleus and cytoplasm, respectively
• RNA modification (including rna methylation)
• Loading onto mRISC
• RNA decay

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10
Q

• Effect of miRNA will depend on:

A

• MiRNA levels
• Target mRNA levels
• Compartmentalisation
• Several miRNAs can target one mRNA

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11
Q

• E.g. Lin-4 : miRNA required for C.elegans development

A

• Lin-4 is a miRNA regulating Lin-14
• Lin-4 If mutants and mutants where the lin-4 binding site in the lin-14 UTR has been mutated (lin-14 gf) fail to turn off lin-14 expression at the L2 stage and both mutants get stuck in larval stage 2
• Lin-4 is required for L1-> L2 transition
• Lin-14 is a target gene of lin-4 and the drop in lin-14 protein caused by lin-4 is required for L1-> L2 transition
• Lin-14 mRNA levels remain constant throughout development
• Lin-4 is an miRNA
• Lin-4 acts by antisense base pairing to 7 complementary elements in the lin-14 mRNA 3-UTR to down-regulate lin-14
• Binding of lin-4 to lin-14 blocks protein synthesis after the initiation of translation
• Absence of this regulation blocks tthe L2 transition to L3

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12
Q
  1. Learn what are operons
A

• Linear sequences of dna
• Clusters of two or more related genes with related functions are under the control of a single promoter and terminator
While ‘operons’ have now been described in eukaryotes here the polycistronic pre-mRNA is co-transcriptionally processed by 3’ end formation and spliced leader trans-splicing between the genes to make mono isotropic mature mRNAs

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13
Q

Parts of an Operon

A

• Promoter – site where RNA pol binds dna
• Terminator – site where rna pol dissociates from dna
• Operator – site where a repressor protein binds to dna
• Repressor – a protein which prevents rna pol binding to dna
• Activator – protein which helps rna pol bind dna
• An Operon is composed of a promoter sequence followed by an operator followed by one or more structural genes (blueprints for proteins)

• Operons are controlled by regulatory genes found elsewhere on the chromosome, which regulate the expression of the structural genes in response to an environmental signal

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14
Q

• Evolution of operons:

A

• Horizontal gene transfer represents one source of operons, and is the primary origin of these functional gene clusters
• Other sources of operon formation/change include:
• ORFan genes (likely from bacteriophages)
• Deletion of genes
• Recombination events bringing more distant genes into proximity
• Gene duplication
• Many operons die when not selected for

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15
Q
  1. Illustrate the structure the prokaryotic RNA polymerase and how it works
A

• Primary channel: accommodates downstream dsDNA and RNA-DNA hybrid
• Secondary channel: site for NTP entry
• Sigma factor sigma70 is responsible for binding of RNApol to general promoters which constitutes majority of genes

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16
Q

Lac operon negative regulation

A

• No lactose – repressor protein (LacI) binds to the operator site and blocks RNA pol
• Negative regulation is relived by lactose
• Lactose binds to LacI, preventing its attachment to the operator and RNApol transcribes DNA

17
Q

Lac operon catabolite repression

A

• Glucose inhibits the activity of the enzyme adenylyl cyclase which catalyses production of cAMP from ATP
• How does glucose inhibit AC: 2 possible theories
• Theory A:
• High glucose -> PTS transfers phosphate to glucose (system saturated) -> PTS(phosphotransferase system) low phosphorylation -> no interaction with adenylyl cyclase -> low cAMP
• Low/no glucose -> little transfer of phosphate groups from PTS to glucose -> phosphorylated PTS proteins accumulate -> phosphorylated PTS interacts wit adenylyl cyclase enhancing its activity -> high cAMP
• Problem is it has also been observed that you can contribute to have a saturated, non-phosphorylate PTS when cAMP rises
• Theory B:
• High glucose -> high ATP (more efficient energy production) -> low adenylyl cyclase and low cAMP -> more anabolism
• Low/no glucose -> low ATP -> high cAMP -> more catabolism to obtain more energy

18
Q

Lac operon consensus sequence

A

• The interactions formed between the CAP and the CAP binding site are:
• ARG180 :: guanine of the consensus base pair G-C at position 5
• Glu 181 :: cytosine of the consensus base pair G-C at position 7
• Arg 185 :: thymine of the consensus base pair A-T at position 8 (and in some structures, also with the guanine of G-C at position 7)

19
Q

Lac operon positive regulation with catabolite repression

A

• Bacteria stop growing at 3 hours due to Catabolite repression
• When glucose is used up there is a lag caused by time taken for operon to be expressed in all bacteria
• Lac operon induction by lactose in single cells is governed by a single-molecule trigger
• Cells that have at least 1 LacZ protein (and also LacY proteins) expressed at the moment of the switch will respond readily
• Cells lacking the lac proteins are unable to sense lactose (sensor less) and have to wait for the next random burst of lac expression (transcriptional noise
• This delays the response to galactose at a population level
• Basal = off

20
Q

Summary of lac operon

A

• Negative regulation:
• LacI repressor : released by lactose
• Catabolite repression:
• Glucose reduces cAMP levels
• CAMP-CAP activates transcription

21
Q

Arabinose operon

A

• AraB, AraA and AraD are required for the breakdown of arabinose (carbon source)
• AraC acts as both an activator and as a repressor
• Negative regulation: AraC binds to O2 and I1
• Positive regulation: AraC binds to I1 and I2
• Catabolite repression: glucose reduces levels of cAMP
• Negative auto regulation: at high levels of AraC, AraC binds to O1 and inhibits its own expression
• When no AraC: more regulatory protein expressed

22
Q

High glucose, low arabinose

A

• When high glucose, low arabinose: negative regulation by AraC
• AraC binds to araO2 and araI to form a loop
• Maximum repression
AraC in this position cannot interact with rna pol bound at promoter so araBAD not transcribed

23
Q

Arabinose operon low glucose, high arabinose

A

When low glucose, high arabinose there is positive regulation by AraC and cAMP-CAP so better expression
AraC binds at AraI1 and AraI2 and interacts with rna pol to permit araBAD transcription

24
Q

• Auto-regulation of AraC expression:

A

• High levels of AraC mean AraC binds to araO1
• RNA polymerase can’t approach ParaC promoter
Suppressed AraC transcription

25
Q

Summary of arabinose operon

A

• Basal=off
• AraC is both activator and repressor
• AraC pBAD from e.coli is one of the more widely used genetic sensor modules in bioengineering because it can drive regulateable and titratable expression of genes and genetic pathways in microbial cell factories

26
Q

Tryptophan operon repression

A

• Anabolic operon so expensive to express
• Negative regulation through Trp repressor (TrpR)
Tryptophan binds to repress or to activate it
Repressor binds to operator

27
Q

Tryptophan operon attenuation

A

Attenuation is only in prokaryotes and couples transcription and translation
• Formation of 2 different stem-loops in the leader mRNA
• Trp low and cell needs Trp ->
• Ribosome stalls at 2 trp codons in region 1 of the leader mRNA
• Stem-loop between 2 and 3 can form which does not affect transcription
• (RNA pol does not stop at attenuator after loop 4)
• Transcription of all trp operon genes trpE-A
• TrpE-A produce tryptophan
• Trp high and cell doesnt need trp ->
• Ribosome quickly translates region 1 and covers region 2
• Stem loop between 3 and 4 can form which terminates transcription
• RNA pol stalls at attenuator, where it dissociates from the U-rich DNA
• No transcription of entire Trp operon genes TrpE-A

28
Q

Tryptophan operon summary

A

• Basal=on
• Total repression by trp repressor and attenuation (700-fold)

29
Q

Riboswitches:

A

Regions of RNA that can bind molecules e.g. FMN
Binding of small molecule to ribsoswitch can trigger formation of hairpin loop that terminates transcription
In others the hairpin loop contains site where ribosome normally binds so interferes with initiation

30
Q

Transcriptional noise

A

• Random bursts of gene expression that are independent of the promoter sequence and its regulation
• Leads to heterogeneity of gene expression among individual bacterial cells
• Even in the same environment individual cells express the same gene differently
• Can be selected for and against

Molecular Biology (9 decks)

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