BIO230 L3 Prokaryotic Transcriptional Regulation

just the names of things

transcriptome analyses

provides a signature of cell type

response to extracellular stimuli

3 steps for RNA processing

5’ capping

RNA splicing

3’ polyadenylation

RNA capping (what is it and 3 functions)

addition of a modified guanine nucleotide to the 5’ end of pre-mRNA (3 enzymes involved)

helps in RNA processing and export from the nucleus

important role in translation of mRNAs in the cytosol (ribosomes bind more efficiently to mRNA w/ 5’ cap)

protects mRNA from degradation (5’ exonucleases will otherwise degrade mRNAs by the 5’ end)

RNA splicing

the process of removing introns (non-coding sequences) using an enzyme complex made up of RNA and proteins called the spliceosome

there are multiple ways to splice RNA termed RNA splicing

sites of proper splicing are bound by EJCs

alternative splicing

different or same cells can produce an RNA transcript differently to make different proteins from the same gene

75% of human genes produce multiple proteins which increase the coding potential of genomes

can be regulated

cap-binding complex (CBC)

binds to cap of mRNA to indicate that it is ready

exon

part of mRNA that is left in — mostly known for being a coding sequence

however there are 2 untranslated regions at 5’ and 3’ called noncoding exons

exon junction complexes

serves as a marker for properly sliced RNA

ribosome knocks off EJC as it translates

if it remains on the mRNA when the ribosome reaches the stop codon, then EJC recruits Upf to degrade mRNA

drosophilia sex determination

involves Sex-lethal, Transformer, and Doublesex which all contain regulated splice sites

females experience a short burst of functional Sxl protein

Sxl protein represses splicing of Sxl and Tra, making functional proteins in females

Tra protein activates splicing of Dsx 

Dsx represses male gene expression, organism undergoes female development

an example of how alternative splicing can be regulated

Sex-lethal (Sxl)

splicing repressor

produces a nonfunctional protein in male drosophilia

a regulator of splicing in females:

protein functions to block splice sites to create functional Tra protein

Transformer (Tra)

splicing activator

produces a nonfunctional protein in male drosophilia

a regulator of splicing in females:

protein functions to activate the splicing of Dsx

Doublesex (Dsx)

regulates gene expression

normally represses female gene expression

represses male gene expression if splicing regulated by Tra and Tra2 protein

3’ polyadenylation and termination

once RNA Pol II reaches the cleavage and poly A signals in DNA, RNA polymerase transfers protein complexes CstF and CPSF to RNA and terminates transcription

CstF and CPSF recruit additional cleavage factors to cleave RNA

poly A polymerase adds ~200 A nucleotides to the 3’ end of RNA from ATP (not genome encoded)

poly A tail is bound by poly-A binding proteins

CstF and CPSF

carried by the C-terminal domain of RNA polymerase II as it transcribes a gene

when RNA pol II reach the poly A signal sequence in the DNA, CstF and CPSF are transferred to the RNA

CstF is the cleavage stimulating factor: helps stimulate cleavage at the correct site 

CPSF provides specificity for recognizing where it should occur

Poly-A polymerase (PAP)

adds ~200 A nucleotides to the 3’ end of RNA from ATP (not genome encoded)

poly-A binding proteins

bind the poly-A tail to aid in

RNA export (required to exit the cell)

translation (important for conformation, interacting with TiF and ribosomes)

mRNA stability (protecting it from degradation by 3’ exonucleases)

tmRNA

acts as both tRNA and mRNA

recruited to the A site of ribosomes processing broken mRNA

carries an alanine amino acid and acts like a tRNA with no anticodon-codon binding

mRNA part is translated by ribosome to create a recognizable tag for degradation

post-translational gene regulation: mRNA stability in prokaryotes

in prokaryotes, exonucleases rapidly degrade most mRNAs

post-translational gene regulation: mRNA stability in eukaryotes

in eukaryotes, mRNAs are more stable and degradation is regulated

2 main mechanisms which both involve gradual poly-A tail shortening like a timer

carried out by deadenylase (an exonuclease) when mRNA reaches the cytoplasm

1. decapping followed by rapid 5’ to 3’ degradation

2. continued 3’ to 5’ degradation

both mechanisms can occur on the same mRNA

cytoplasmic poly-A elongation can also occur to stabilize mRNA

proteins can also interfere with poly-A shortening

mRNA stability: transferrin receptor

transferrin receptor imports iron into the cell when iron is low

in iron starvation, mRNA is stabilized by cytosolic aconitase binding the 3’ UTR region (where poly A tail is) to make a hairpin structure

in excess iron, aconitase binds iron and releases mRNA, exposes the 3’ UTR endonucleolytic site, causing rapid degradation of mRNA

aconitase

helps regulate transcription of transferrin receptor by binding to its 3’ UTR in low iron concentration and releasing the mRNA to be destroyed when bound to iron in high iron environments

mRNA stability: deadenylase

competition between mRNA translation and mRNA degradation

deadenylase shortens the poly-A tail binds the 5’ cap like eIFs

when transcription initiation factors are bound to the 5’ cap and poly-A tail, deadenylase cannot bind and vice versa

microRNAs (miRNAs)

non-coding RNAs that base pair with specific mRNAs to regulate mRNA stability

initially double-stranded, has a 5’ cap and poly-A tail, is “cropped” to remove these features

is “diced” to become single stranded and bound with Argonaute

after special processing, it associates with RNA-induced silencing complex (RISC)

if an extensive match is formed, the target mRNA is sliced in half and degraded

RNA-induced silencing complex (RISC)

seeks mRNA with complementary nucleotide sequences. two outcomes of base pairing are possible.

1. extensive match: target undergoes SLICING and RISC is phosphorylated to un-pair, leading to RAPID DEGRADATION

2. less extensive match: target undergoes rapid translational REPRESSION, DEADENYLATION, and mostly EVENTUAL DEGRADATION

RNA interference (RNAi)

RNAi destroys double stranded RNA

initiated by dicer protein complex

small interfering RNAs (siRNAs) can interact with Argonaute and RISC proteins follow the miRNA route to destroy double-stranded RNA 

siRNAs can also regulate transcription by interacting with Argonaute and the RNA-induced transcriptional silencing (RITS) complex

found in eukaryotes

RNA-induced transcriptional silencing complex (RITS)

interacts with newly transcribed RNA 

recruits chromatin modifying enzymes to repress transcription

histone methylation, DNA methylation, transcriptional repression (similar to how chromatin is used in DNA to suppress genes)

Argonaute

a protein of RISC that plays a critical role in base-pairing miRNA with mRNA

CRISPR-Cas immunity

an immune reaction in prokaryotes

short fragments of viral DNA integrate into the CRISPR region of the genome and become templates to produce crRNAs

viral DNAs complementary to CRISPR regions are directed for degradation by Cas proteins

similarly to Argonaute: use of small single-stranded RNA

CRISPR

a region of the prokaryotic genome where viral DNA is collected to become crRNAs

viral DNAs complementary to CRISPR regions are degraded

CRISPR-associated proteins (Cas)

directs viral DNA complementary to CRISPR regions to degradation

Lac repressor

binds to the lac operator to block RNA polymerase through competitive binding

unbound when lactose (ligand) concentration is high

example of negative regulation (bound repressor prevents transcription)

catabolic activator protein (CAP)

binds to the CAP binding site to recruit RNA polymerase

bound when cAMP (ligand) concentration is high due to low glucose

example of positive regulation (bound activator promotes transcription)

Trp repressor

binds to the Trp operon to block RNA polymerase through competitive binding

bound when tryptophan (ligand) concentration is high

contains helix-turn-helix DNA binding motif

example of negative regulation (bound repressor prevents transcription)

helix-turn-helix 

DNA binding motif where protein regulator binds in the major groove of the DNA double helix

negative regulation

competition between RNA polymerase and repressor protein for promoter binding

positive regulation

activator protein recruits RNA polymerase to the promoter to activate transcription

gene regulatory elements are typically found (prokaryotes, eukaryotes)

prokaryotes

• close to the transcriptional start site

eukaryotes

• far upstream of gene

• downstream of gene

• within gene (introns)

NtrC protein

transcriptional activator located far from promoter

DNA looping allows NtrC to directly interact with RNA polymerase to activate transcription from a distance

bacteriophage lambda

virus that infects bacterial cells

example of positive and negative regulatory mechanisms work to regulate the lifestyles of bacteriophage lambda

two gene regulatory proteins are responsible for initiating this switch and repress each other’s synthesis

lambda repressor protein (cI)

represses lysis and activates prophage in bacteriophage lambda

occupies the shared operator, blocks synthesis of Cro

activates its own synthesis by recruiting RNA polymerase, most bacteriophage DNA not transcribed

positive feedback loop

Cro

represses prophage and allows lysis in bacteriophage lambda

occupies the shared operator, blocks synthesis of lambda repressor

most bacteriophage DNA extensively transcribed

DNA replicated, packaged, new bacteriophage released by host cell lysis

flip-flop device (indirect positive feedback loop)

conditions to trigger the switch between prophage and lysis in lambda bacteriophage

host response to DNA damage — switch to lytic state

• inactivates repressor

good growth conditions — maintains prophage state

• lambda repressor turns off Cro and activates itself in a positive feedback loop

positive feedback loop (uses)

e.g. lambda repressor protein

can be used to create cell memory — like staying differentiated in cell division

negative feedback loop

more A turns off itself

flip flop device (indirect positive feedback loop)

e.g. Cro/repressor switch

feed-forward loop (uses)

two gene products required to regulate a third

can measure the duration of a signal

how positive feedback loops can be used to create cell memory

proteins remain in cell after division

how feed forward loops can measure the duration of a signal

relies on accumulation of protein

the repressilator

simple gene oscillator using a delayed negative feedback circuit

however amplitude increased over time due to bacterial growth

an example of synthetic biology

circadian gene regulation in drosophila

uses delayed negative feedback loop to regulate sleep cycle

transcription attenuation

RNA adopts a structure that interferes with RNA polymerase causing a premature termination of transcription

regulatory proteins can bind to RNA and interfere with attenuation

prokaryotes, plants, some fungi use riboswitches to regulate gene expression

riboswitches

short RNA sequences that change conformation when bound by a small molecule

prokaryotes, plants, and some fungi use riboswitches to regulate gene expression

eg prokaryotic riboswitch that regulates purine biosynthesis

prokaryotic riboswitch that regulates purine biosynthesis

low guanine levels activate transcription of purine biosynthetic genes

high guanine levels cause guanine to bind to riboswitch and RNA polymerase to terminate transcription of purine biosynthetic genes

TFIID

general transcription factor

recognizes TATA box and other DNA sequences (other consensus sequences) near the transcription start point for initiation

bends DNA to highlight it for RNA polymerase recognition

TFIIH 

general transcription factor

unwinds DNA at the transcription start point 

phosphorylates Ser5 of the RNA polymerase C-terminal domain (CTD)

releases RNA polymerase from the promoter to initiate transcription

RNA polymerase II

transcribes protein coding genes in eukaryotes

general transcription factors

TFIID, TFIIB, TFIIF, TFIIE, TFIIH for eukaryotes

sigma factor for prokaryotes

difference between eukaryotic genomes and prokaryotic genomes

eukaryotic genomes

• lack operons

• 1 gene, 1 RNA molecule, 1 protein

• packaged into chromatin (additional mode of regulation)

prokaryotic genomes

• operons 

• multiple genes encoded on 1 RNA molecule

mediator

acts as an intermediate between many regulatory proteins and RNA polymerase in eukaryotes

coactivators and corepressors

only bind to proteins that are already bound to DNA, do not directly bind to DNA

DNA binding domain (DBD)

a module of eukaryotic activator protein that recognizes specific DNA sequence

very specific about binding

activation domain (AD)

a module of eukaryotic activator protein that accelerates frequency/rate of transcription

tends to be general

how activator proteins activate transcription

attract, position, and/or modify general transcription factors, mediator, RNA polymerase II

by either directly interacting with them, or indirectly modifying chromatin structure

activator proteins can bind directly to transcriptional machinery or the mediator and attract them to promoters

nucleosomes

the basic structure of eukaryotic chromatin

DNA wound around a histone octamer with 2 sets of H2A, H2B, H3, H4 creates a compact chromatin fibre that can be unwound or altered to increase promoter accessibility

4 major ways activator proteins can alter chromatin

chromatin remodeling complex:

• nucleosome sliding

• histone removal

• histone replacement

histone-modifying enzyme:

• specific pattern of histone modification