Genetics Exam 3

eukaryotic translation initiation factors

• eIF1A: blocks the A site

• eIF1: prevents premature interaction; keeps 60S (large subunit) away from 40S (small subunit)

• eIF2: brings the first (initiator) tRNA

  • in eukaryotes, the initiator tRNA comes in before the mRNA

• eIF3: prevents premature interaction; keeps 60S away from 40S

• eIF5: brings the initiator tRNA

• eIF5B: promotes binding between 60S and 40S to form the mature 80S ribosome

capping complex eIF4F

escorts mRNA to the ribosome and includes:

• eIF4A: has helicase activity and ATPase activity

  • ATPase activity allows scanning: to find the start codon, the 40S (small subunit) is pushed along the transcript to find the start codon (the Kozak sequence)

• eIF4G: interacts with the poly (A) tail

  • poly (A) binding proteins (PABPs) bind to the poly (A) tail

  • G binds to PABPs to circularize the mRNA, which allows us to keep making the same protein

• eIF4E: binds to the 5’ cap

Kozak sequence

a specific nucleotide sequence found in eukaryotic mRNA that surrounds the AUG start codon, allowing the ribosome to recognize and initiate translation efficiently

5’ GCCGCCRCCAUGG 3’ where R is any purine (A or G) and AUG is the start codon

eukaryotic translation elongation factors

eEF1⍺: brings in the incoming tRNA

eEF2: translocation

eukaryotic translation termination factors

eRF1: recognizes all three stop codons (UAA, UAG, UGA)

eRF3: brings eRF1 to the A site

polycistronic mRNA

an mRNA that can code for several proteins (prokaryotic)

monocistronic mRNA

an mRNA that can only code for one protein (eukaryotic)

operon

a functional unit of DNA that consists of related genes under the influence of one promoter

• encodes a polycistronic mRNA (one long piece of mRNA that we can process into several genes)

• this allows a bacterium to coordinately regulate a group of genes that encode proteins with a common functional goal

important DNA sequences in the operon

• promoter: where RNA polymerase binds to initiate transcription

• operator: located between the promoter and structural genes; binds a repressor protein to regulate transcription

• structural genes: a sequence of genes that are co-regulated and transcribed together

• terminator: signals the end of transcription

inducible operon

usually inactive (OFF) because a repressor is bound to the operator. it is turned on (induced) when a specific small molecule (inducer, usually the metabolite) binds to the repressor, inactivating it and allowing transcription to proceed (ex. lac operon)

repressible operon

usually active (ON) because the repressor is initially inactive. it is turned off (repressed) when a molecule (co-repressor, usually the metabolite) binds to the repressor, activating it to bind to the operator and stop transcription (ex. trp operon)

attenuation

premature termination of transcription occurs; the cell starts transcription but cuts it short

• ex. high tryptophan levels in E. coli lead to a terminator hairpin structure that stops transcription early, while low tryptophan levels allow transcription to continue

repressor

a regulatory protein that binds to the operator and inhibits transcription (negative control)

activator

a regulatory protein that binds to the activator binding site and increases transcription (positive control)

enzyme adaptation

a particular enzyme appears in the cell only after the cell has been exposed to the enzyme’s substrate

• if we grow cells in the presence of lactose, there is an increase in the enzyme needed to break down lactose

DNA elements of the lac operon

• promoter: binds RNA polymerase (lacP)

• operator: binds the lac repressor protein (lacO)

• CAP site: binds the catabolic activator protein (CAP)

  • needs cAMP: allosteric molecule that promotes CAP to bind to CAP site

structural genes of the lac operon

• lacZ: encodes β-galactosidase (enzyme that breaks down lactose into glucose and galactose)

  • cleaves lactose and lactose analogs

  • converts lactose to allolactose

• lacY: encodes lactose permease (membrane protein required for transport of lactose into cells)

  • creates channels that allow lactose to pass into the cell

• lacA: encodes galactosidase transacetylase (transfers an acetyl group from one molecule to another)

  • covalently modifies lactose and analogs

  • prevents toxic buildup by adding an acetyl group to lactose to change its structure and make it useless ← too much lactose is bad for the cell

the lacI gene

• encodes the lac repressor

• not considered part of the lac operon; has its own promoter (the i promoter)

• always expressed, so we will always make the repressor (constitutive gene)

diauxic growth

growing cells in the presence of glucose and lactose

• glucose ↓ cAMP → no CAP activation → lac operon OFF

• when glucose is depleted → ↑ cAMP + lactose inactivates repressor
  → lac operon ON → second growth phase

enzymes involved in tryptophan biosynthesis

trpE, trpD, trpC, trpB, trpA

genes involved in trp operon regulation

• trpR: encodes the trp repressor protein

  • functions in repression

• trpL: encodes a short peptide called the leader peptide

  • functions in attenuation

    • region 1 contains two consecutive tryptophan codons (UGGUGG), which act as the sensor for tryptophan concentration

      • high concentration → the ribosome moves quickly through the region 1 codons, allowing region 2 to pair with region 3, forming a terminator stem-loop

      • low concentration → the ribosome stalls at the region 1 codons because it cannot find the necessary tRNA; this allows the antiterminator to form, which allows RNA polymerase to continue transcribing the operon

transcription factors

proteins that influence the ability of RNA polymerase to transcribe a given gene; two main types

general transcription factors

required for the binding of RNAP to the core promoter and its progression to the elongation stage; lure RNAP

• TFIID

• TBP (TATA-box binding protein)

regulatory transcription factors

• regulate the rate of transcription of target genes and influence the ability of RNAP to begin transcription of a particular gene; influence RNAP to go fast, slow, etc.

• recognize regulatory elements located near the core promoter; binding of RTFs to regulatory elements affects the transcription of an associated gene

activator

a regulatory protein that increases the rate of transcription

• the sequence it binds to is called an enhancer

repressor

a regulatory protein that decreases the rate of transcription

• the sequence it binds to is called a silencer

up-regulation

the binding of a transcription factor to an enhancer increases the rate of transcription 10-1000 fold

down-regulation

the binding of a transcription factor to a silencer decreases the rate of transcription

many regulatory elements are orientation-independent/bidirectional

can function in the forward or reverse direction; most enhancers/silencers are upstream of the promoter but some can be downstream

• proximal enhancer/silencer: located near the promoter

• distal enhancer/silencer: located far away from the promoter

domains

regions in transcription factor proteins that have specific functions (e.g., one domain for DNA binding, one domain for allosteric/effector/signaling molecules to bind)

relaying the message from RTFs → RNAP

most RTFs do not bind directly to RNAP; they bind to DNA sites and their effects are communicated through three common interactions (regulation via TFIID, regulation via mediator, chromatin remodeling)

transcriptional activation via TFIID

the activator/coactivator complex recruits TFIID to the core promoter and/or activates its function → transcription will be enhanced

transcriptional repression via TFIID

the repressor inhibits the binding of TFIID to the core promoter or inhibits its function → transcription is silenced

transcriptional activator via mediator

• the activator protein interacts with mediator, resulting in the phosphorylation of the carboxyl-terminal domain (CTD) of RNAP

• some GTFs are released, and RNAP proceeds to the elongation phase

transcriptional repression via mediator

the repressor interacts with mediator in a way that prevents the phosphorylation of the CTD, so elongation cannot proceed

ATP-dependent chromatin remodeling

dynamic changes in chromatin structure that range from a few nucleosomes to large scale changes; to move things around so binding sites become available

• energy of ATP hydrolysis is used to drive change in location and/or composition of nucelosomes

• DNA translocase: creates space for factors to bind

eukaryotes have multiple families of chromatin remodelers

• SWI/SNF: known for opening chromatin, sliding, and ejecting nucleosomes, which generally activates gene expression

• ISWI: primarily involved in nucleosome spacing, assembling regular nucleosomal arrays, and chromatin compaction; function as a "ruler" to determine linker DNA length

• INO80: specialized in exchanging histone variants (e.g., replacing H2A with H2A.Z) within the nucleosome; involved in DNA damage repair

• Mi-2: coupled activity of nucleosome sliding and histone deacetylase (HDAC) activity, typically leading to chromatin compaction and gene silencing

histones

highly basic proteins (made up of histidine, lysine, and arginine) with an octamer core:

• 2 H2A, 2 H2B, 2 H3, and 2 H4

• H1 binds to the DNA linker region between nucleosomes, helping to tighten the wrapping and condense chromatin

• histone tails: N- or C-terminal amino acid extensions of histone proteins that protrude from the nucleosome core

chromatin structure

• closed conformation: chromatin is very tightly packed; some nucleosome-free regions but transcription may be difficult or impossible

• open conformation: chromatin is accessible to transcription factors; transcription factors

three types of chromatin remodeling

• change in the position of nucleosomes (relative positions or spacing over a long distance)

• eviction of histone octamers

• replacement with histone variants