Transcription Flashcards
transcription
- dna to rna
- proceeds in 5’ to 3’ direction
- template strand read from 3’ to 5’ direction
- nontemplate strand is coding strand
- rna polymerase can only work on one strand at a time
- RNA polymerase doesn’t know which strand is which, just reads in 3’ to 5’
parts of a gene from left to right(5’ to 3’)
promotor -> transcription start -> translation start -> open reading frame-> translation stop -> transcription stop
promoter
- serves as recognition site for binding RNA polymerase
- -contains regions rich in A and T
- closer promoter corresponds to consensus sequence, greater its strength
- strong promoters bind RNA polymerase more tightly, frequently, and successfully, so greater efficiency
- occurs on nontemplate strand
- diff promoters specify which tissues express diff genes
- diff promoters specify when different genes are expressed during development (grey hair, hormone expression, etc)
consensus sequence
identify region as promotor
E. coli RNA polymerase B’ subunit
DNA binding
E. coli B subunit
catalytic site
E. coli alpha subunit
promoter binding, assembly, and regulation
E. coli w subunit
structural role, restores activity
sigma Factor in E. Coli RNA polymerase
-promoter recognition
initiation and elongation stages of transcription in prokaryotes
- RNA polymerase binds to promoter, causing strand separation and unwinding
- negative and positive supercoils form on each side of RNA polymerase as DNA unwinds; topoisomerase relieve stress
- initiation is complete after 10 NTPs have been added
- elongation continues as sigma factor falls away
lamp brush chromosomes
- in eukaryotes
- once enough space on DNA template has been freed, 2nd, 3rd, etc additional RNA polymerases attach behind first to form multiple copies of RNA
termination of transcription: Rho factor
- an ATP dependent helicase that catalyzes the unwinding of RNA-DNA duplex hybrids during transcription to promote termination of prokaryotic transcription
- attaches to transcript and follows RNA polymerase
- hybrid duplex is unwound, RNA is detached when polymerase “stalls” at the terminator sequence
termination of transcription: prokaryotes
- factor independent termination
- RNA polymerase reaches terminator regions of DNA
- Terminator: Poly A regions of DNA that code for “hairpin” mRNA structures
- mRNA and polymerase fall away
hairpins
contract length of message, complementarity is lost, transcription complex is destabilized
areas of complexity in eukaryotic transcription
- 3 different RNA polymerases-none able to initiate transcription
- promoters are more complex with more consensus sequences
- initiation requires many “transcription factors” to activate RNA polymerase
- regulatory elements (enhancer, silencers) modify gene expression
- transcripts require considerable processing prior to translation
RNA polymerase I
- located in nucleolus
- transcribes large rRNAs
RNA polymerase II
- located in nucleus
- transcribes mRNAs, snRNAs
- interacts with several transcription factors: TATA box binding protein, and others
RNA polymerase III
- located in nucleus
- transcribes tRNAs
- 5s rRNAs
eukaryotic RNA polymerases
CANNOT initiate transcription
housekeeping gene
codes for proteins needed all the time
initiation of eukaryotic transcription
- involves several transcription factors
- transcription factors sequentially bind to TATA region and polymerase
- polymerase complex binds to promoter
- TFIIH (transcription factor II H) activates polymerase via phosphorylation and transcription begins
enhancers
- short segments of DNA near eukaryotic promoters that bind transcription factors to enhance the level of transcription of certain genes
- formation of DNA loop allows interaction (activates) with RNA polymerase
- communication between enhancer regions and proteins bound at promoter proceed through multiprotein complex called mediator
termination of transcription in eukaryotes
- RNA polymerase II usually transcribes past end of gene
- pre-mRNA carrying AAUAA signal is cleaved 11 to 30 residues downstream of these sites
- polyA tail is then added by polyA polymerase
- polyA tails relate to mRNA stability, the longer the tail, the longer the half life
mRNA polyadenylation
- addition of a polyA tail to the 3’ end of mRNA transcripts after AAUAA termination sequence
- helps direct mRNA’s out of nucleus to cytoplasm
- protects 3’ end from exonuclease degradation
- length of tail is related to longevity of transcript
- polyA polymerase is required for polyadenylation
RNA processing
- transcription involves synthesis of several RNAs
- different RNA species are in different stages of “completion”
- many require further modification (processing) before they can be used
rRNA and tRNA processing in prokaryotes and eukaryotes
- rRNAs and tRNAs are encoded by operons
- Pre-RNAs must be cut into appropriate segments by an assortment of specific RNases
- prokaryotic mRNAs don’t require processing
mRNA processing in eukaryotes
- 5’ capping - addition of 7-methyl guanosine to 5’ end
- 3’-polyadenylation-addition of polyA tail to 3’ end
- splicing- the removal of introns and ligation of exons
polyadenylation
helps direct mRNAs out of nucleus to the cytoplasm
-protects 3’ end from exonuclease degradation
5’ cap of eukaryotic mRNA
- methylguanosine linked via 5’ to 5’ triphosphate (stable)
- 2’ OHs of up to 3 nucleotides are methylated
- 5’ cap serves as a recognition site for ribosome attachment and prevents transcript degradation by exonucleases
exons
coding regions in eukaryotic genes
introns
non-coding regions in eukaryotic genes
mRNA splicing
- non-coding regions of pre-mRNAs must be excised and coding regions ligated together to form functional mRNA
- spliceosome formed by snRNPS joining together
- spliceosome cuts out introns and attaches exon ends together
- occurs in nucleus
spliceosome
- snRNP-pre-mRNA complex in mRNA splicing
- may catalyze splicing
possible functions of introns
- exon shuffling
- signal for mRNA export from nucleus
positive control
genes are not transcribed unless an activator is present
activation
turns gene on
deactivation
turns gene off
negative control
genes are always transcribed UNLESS a repressor is present
repression
turns gene off
derepression
turns gene on
operons
groups of genes with related functions where a single promoter and operator save to control expression of all genes in that unit together
lac operon
- made up of lacZ, lacY, and LacA
- control lactose utilization in E. coli
- when transcribed, operon yields polycistronic mRNA (multiple coding sequences exist)
constitutive phenotypes
all 3 genes are synthesized at high levels, even in the absence of inducer
noniducible phenotypes
all 3 gene activities remain low, even after addition of inducer
structures of lac operon inducers
–allolactose is true intracellular inducer
repression in lac operon
repressor protein synthesized by LacI gene binds a specific sequence in operator and blocks transcription by preventing RNA polymerase from binding
Derepression (activation) of lac operon
- repressor has inducer binding site
- when repressor binds inducer, affinity of repressor for operator DNA is greatly reduced, derepressing transcription
- provides negative control of lac operon
transcriptional activation of lac operon when glucose levels are low
- in E. coli, when glucose levels drop, cAMP levels rise
- cAMP interacts with cAMP receptor protein, activating lac operon
- binding of cAMP to CRP causes conformational change, increasing affinity for DNA
- CRP helps recruit RNA polymerase, stimulating transcription
- E. coli like to metabolize glucose because it’s a monosaccharide so easier to break down
transcription regulation in eukaryotes
- more complex than prokaryotes
- eukaryotic genome contains a large number of transcription factors
- regulation by TFs is combinatorial
- complexity w multicellularity demands higher order of regulation
- eukaryotes use chromatin (not naked DNA) as the template
- complexity brought about by small regulatory RNA molecules
modes of gene regulation in eukaryotes
- genomic control
- rna processing
- regulation of nuclear RNA export out of nucleus
- translational control
- signal transduction
heterochromatin
condensed, darkly stained chromosomal DNA in nuclei
euchromatin
expanded, lightly stained chromosomal DNA in nuclei
cytosine methylation
- inhibits RNA polymerase
- silences expression
- mostly seen in heterochromatin
acetylation of histone lysines
- promotes formation of euchromatin
- more accessible for transcription
high levels of acetylation = ?
high transcriptional activity
bromodomains
- -ATPase domain
- interact with acetylated lysine residues
- uses energy from ATP hydrolysis to couple energy to modify histone
chromodomains
- ATPase domain
- interact with methylated histones
- uses energy from ATP hydrolysis to couple energy to modify histone
coffin-lowry syndrome
- histone modification syndrome
- mental retardation and abnormalities of the head and facial and other areas
- caused by mutations in the RSK2 gene (histone phosphorlylation)
- inherited as X-linked dominant genetic trait
- Males usually more severely affected
Rubinstein-Taybi Syndrome
- histone modification syndrome
- characterized by short stature, intellectual disability, distinctive facial features, broad thumbs and 1st toes
- caused by mutation in CREB-binding protein (histone acetylation)
DNA methylation in eukaryotes
- methylation patterns altered in cancer
- only base methylated is cytosine
- methylation occurs as cytosine residues
- CpG regions typically underrepresented
- Methylation patterns are heritable
- mammalian cells possess 3 different DNA methyltransferases (DNMTs)
- can lead to permanent gene inactivation
- not one single mechanism accounting for gene repression from methylation
Dmnt1
responsible for maintenance of methylation patterns
DNA CpG methylation in eukaryotes
-responsible for inactivation of X chromosome and gene imprinting
Prader Willi Syndrome
- DNA methylation syndrome
- characterized by learning difficulties, short stature, compulsive eating
- individual missing paternal gene activity or missing activity in 2 maternal gene copies
Angelman Syndrome
- DNA methylation syndrome
- characterized by learning difficulties, speech problems, seizures, jerky movements, unusually happy disposition
- individuals are missing maternal gene activity or missing activity from two paternal genes
cAMP mediated signal transduction
- hormone binds to membrane recpetor
- GTP replaces GDP on inactive G protein/membrane
- this converts G protein to active form
- Active GTP-G protein complex activates adenylate cyclase
- adenylate cyclase produces cAMP
- cAMP activates protein kinase
- protein kinase phosphorylates an inactive enzyme to convert to active form
