Gene Expression Flashcards by Kayla Ferguson

What are constitutive genes?

genes that are expressed all the time

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What are inducible genes?

genes that are turned on at specific times or after certain cues

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Most of the regulation occurs at what step?

initiation of transcription (happens at a lot of steps)

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How is the heat shock gene in E. coli regulated?

have variations in core promoter sequence, they recruit a different sigma

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What does the normal E. coli promoter vs the heat shock promoter bind?

normal binds sigma70, heat shock binds sigma32

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How does negative regulation happen?

through a repressor, binds and restricts access of promoter to rna polymerase

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How does positive regulation happen?

through an activator, helps recruit rna polymerase

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What enzyme is required for the breakdown of lactose?

beta-galactosidase

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What do operons typically consist of?

gene of interest, promoter (RNA pol binding site), enhancers, operators

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What do enhancers do?

binding site for transcriptional activators

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What do operators do?

binding site for transcriptional repressors

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What is +1?

regulatory sites are in close proxmitity to start site of transcription are called this

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What does the lac repressor do in the absence of lactose?

forms a homotetramer and binds to either the O3 and O1 sites or the O2 and O1 sites (these are operators), stops rna polymerase from binding

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What is the inducer of the lac operon?

allolactose, IPTG is the synthetic inducer

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How is allolactose made? How does it do its job?

lactose is converted into allolactose by LacZ at low levels, binds Lac repressor (Lacl) to make it let go of operator

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How is the lac operon regulated through positive regulation?

the cAMP receptor protein binds near the promoter when it is bound to cAMP, recruits rna polymerase

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When and what makes cAMP?

made by adenylate cyclase when glucose levels are low

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What are riboswitches?

metabolite sensing in the 5-untranslated regions on mRNAs, change shape which influences gene expression

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Genes containing riboswitches typically code for what?

proteins that are involved in the syntehsis of molecules that are expensive to produce, TPP, FMN etc

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What are riboswitches composed of?

aptamer (binds metabolite) and expression platform (expression regulator)

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How has TPP been shown to alter mRNA structure to inhibit its expression?

creates transcriptional terminator
changes structure so ribosome binding site it blocked
alters availibility of splicing zones for mRNA will be non functional

What is alternative splicing?

joining of different combinations of exons to form cell-specific proteins

What does alternative splicing allow?

1 gene to make multiple different proteins

What is alternative polyadenylation signals?

two different cleavage and polyadenylation sites can be used to make different messages and different proteins

What is rna interference?

cellular mechanism that uses the gene's own DNA sequence to turn it off

What is the first step of rna interference?

rna pol ii makes noncoding pri -miRNA that are processed by Drosha microprocessor complex into pre-miRNA

In rna interference what happens after pre-miRNA is made?

exported to cytoplasm by exportin 5 and processed into double stranded rnas (dsRNAs) by Dicer

What happens after dsRNAs are made in RNAi?

loaded into RNA induced silencing complex (RISC), one strand is kept (guide) and other is degraded (passenger)

After the passenger strand is degraded waht happens next in RNAi?

the guide strand is used by RISC to bind mRNA's by complementarity, if its complenetary than mRNA is cleaved and degraded, if partially complementary then translation repression

What are plasmids?

naturally occurring, extra-chromosomal pieces of DNA that contain all the necessary DNA sequences to replicate autonomously in a bacterial host

Where is the DNA of interest inserted into plasmid?

restriction site

What does the insert vs the vector mean?

the dna that we inserted, the plasmid

What does every plasmid need?

origin of replication, antibiotic resistance marker, restriction enzyme sites for the specific cleavage of the plasmid dna to put into the insert

What does the antibacterial marker do?

allow bacterial cells that contain the plasmid be selected from cells that do not

What do they believe restriction endonucleases evolved as?

defense mechanism against foreign dna

What are the two ways a restriction enzyme can cut? What is the result?

staggered, sticky end same place, blunt end

What is the likelihood that a 6 nucleotide long restriction enzyme site is found within a random DNA sequence?

1 in 4096 (4^6)

Most plasmids used for cloning are engineered to contain what?

a polylinker that contains several recognition sequences for commonly used enzymes

What can expression vectors do?

used to express whatever gene of interest you insert into the polylinker

What do expression vectors contain?

bacterial promoter and ribosome binding elements from whatever DNA is inserted into the plasmid

What is the first step of PCR?

strand is heated to 95 degrees to seperate the strands

What comes after the denature step in PCR?

oligonucleotide primers are added, cooled so they can anneal (50 degree), add Taq DNA polymerase

Why Taw dna polymerase in PCR?

can withstand higher temperatures without denaturing

What is cDNA?

dna that is complementary to the mature mRNA (has introns removed)

How are cDNAs generated?

reverse transcriptase, converts RNA to DNA

Once one cDNA is made how can we get more? What is this called?

PCR steps, RT-PCR

How does sanger sequencing work?

primer is annealed to a known sequence upstream of the unkown sequence, then polyermase will add nucleotides until it reaches a chain terminator, can then use electrophoresis to read the segment

What are chain terminators?

dideoxy nucleotides, has no free OH so nothing can be added after it

What is illumina sequencing?

similar to sanger sequencing but instead of ddNTPs we use dNTPs that are fluorescent different colours

If growing E. coli in a high lactose and high glucose environment what do you expect to find at promoter?

No lax repressor bound to operator and no cAMP bound to activatir