Basic Transcription Part 1 Flashcards
three steps in transcription
- initiation - rate-limiting
- elongation
- termination
differential gene expression
- all cells in your body have the same DNA but what makes cells unique are the genes that are expressed in them
transcription of genes
- one part of more general term of gene expression
Coding Strand
- sequence that resembles mRNA
Template Strand
- make transcript
- read by RNA pol
RNA transcription requires
- RNA polymerase enzymes
- promoter sequence on the DNA template
- ribonucleotides
holoenzyme
- entire enzyme
- core + sigma factor
core
- all but sigma factor
- Beta and Beta’
- alpha
sigma factor
- specificity factor
- directs the polymerase to initiate transcription at specific promoters
sigma promotes what type of binding
- tight binding between RNA pol and promoters
results of filter binding assay
- holoenzyme dissociates with a half time of 30-60 hours
- core dissociates with a half time of less than 1 minute
- holoenzyme binds tightly
holoenzyme binding to promoters
- tight because of sigma subunit
holoenzyme binding to DNA
- loose
+1
- first nucleotide incorporated into RNA
adding a nucleotide to RNA
- nucleic acids always added to 3’ OH
- attack of old 3’ OH to new 5’ PO4
attack of old 3’ OH to new 5’ PO4
- release of pyrophosphate and formation of phosphodiester linkage
Does RNA polymerase require a primer
- No
Does DNA polymerase require a primer
- 3’ OH primer
Promoters
- DNA sequences where RNA polymerases bind to initiate transcription
- Major points of control of gene regulation
Prokaryotic RNA polymerases
- have the intrinsic capacity to recognize promoters
Eukaryotic RNA polymerases
- do not have the intrinsic capacity to recognize promoters
- need a number of other protein factors to bind near promoters to recruit RNA pol
(-)10 and (-)35 sequences
- sequences are recognized and bound by sigma subunit of RNA pol
- space needed for RNA pol to bind
prokaryotic promoter composed of
- upstream element
- Fis sites
- core promoter
upstream element
- promotes high levels of transcription
- stabilize interaction with pol and core promoter
nucleotides upstream of the +1
- are not transcribed.
regions of the sigma factor
- regions 1-4
region 1
- found only in primary sigma factor (found in all cells all the time)
- prevents sigma from binding DNA by itself
region 2
- most highly conserved
- binds core
- promoter recognition at -10
region 3
- helix-turn-helix core and DNA binding
region 4
- promoter recognition of -35 box
regions of sigma region 2
- Regions 2.1
- Regions 2.4
Regions 2.1
- binds core
- amino terminus
Regions 2.4
- promoter recognition at -10 box
sigma cycle
- binds to RNA pol
- transcribes DNA
- falls off
- recycled again
terminators
- at end of gene cause polymerase to fall off template releasing template
two types of terminators
- intrinsic terminators - requires no proteins
- RNA dependent terminators - requires Rho
intrinsic termination process
- hairpin begins to form due to string of Ts
- hairpin destabilizes the RNA-DNA hybrid which helps keep RNA pol on DNA
- transcript falls out
- polymerase falls off
rho dependent termination
- requires hairpin loop but no string of T’s
- Rho binds to transcript at rho loading site and pursues polymerase
- hairpin forms, polymerase pauses, rho catches up
- rho helicase releases transcript by unwinding DNA-RNA hybrid
- causes termination
