Chapter 8: Transcription Flashcards
the copying of one strand of the DNA (the coding strand) into an RNA molecule (transcript)
transcription
enzyme that transcribes DNA into RNA
RNA polymerase
RNA polymerase copies the coding strand of DNA by using the ()
complementary (template/non-coding) strand
RNA polymerase separates the DNA strands and allows () to base pair with template strand
ribonucleoside triphosphates
types of RNAs that are produced
- mRNA (encode proteins)
- non-coding RNAs (regulatory, catalytic, or structural roles)
3 stages of transcription
- initiation
- elongation
- termination
transcription starts when RNA polymerase binds to DNA sequence just preceding the gene: (); signifies start site for transcription
promoter
first base to be transcribed; denoted +1
transcription start site (TSS)
if RNA is transcribed in the 5’ to 3’ direction, the template must be read in the () direction
3’ to 5’
on the RNA, bases 3’ of a site are (1), while bases 5’ of a site are (2)
- downstream
- upstream
during initiation, RNA polymerase separates DNA strands to make a ()
transcription bubble
during initiation, the first few ribonucleoside triphosphates are added while ()
RNA polymerase is still at promoter
RNA polymerase moves past promoter and changes conformation to be more stably associated with DNA when RNA is ()
a sufficient size
transcription stage where RNA polymerase moves along the DNA, adding ribonucleotides
elongation
how is the transcription bubble maintained during transcription elongation
DNA re-pairs behind RNA polymerase as the enzyme unwinds DNA ahead
elongation continues until the polymerase meets a DNA sequence called a () that signals RNA synthesis to cease
terminator
in chromatin-packaged DNA, () prevent transcription machinery binding to DNA
nucleosomes
3 additional types of enzymes required in eukaryotic transcription
- nucleosome remodeling enzymes
- histone chaperones
- enzymes that reversibly modify histone proteins
enzymes that reposition histones away from DNA to be transcribed; can also work to block transcription
nucleosome remodeling enzymes
enzymes that disassemble and reassemble the histone octamer
histone chaperone
some enzymes can reversibly modify histone proteins; these modified proteins ()
recruit specific proteins to certain DNA regions
eukaryotes have 3 RNA polymerases
RNA polymerase I to III
eukaryotic RNA polymerase that transcribes large ribosomal RNA (rRNA) genes
RNA Pol I
eukaryotic RNA polymerase that transcribes messenger RNA (mRNA) genes
RNA Pol II
eukaryotic RNA polymerase that transcribes a variety of RNAs including transfer RNAs (tRNAs) and 5S ribosomal RNA
RNA Pol III
() have a 4th RNA polymerase that transcribes regulatory RNAs
plants
number of polymerases in bacteria and archaea
1
all RNA polymerases have a core enzyme that (1), but this cannot act alone and relies on extra proteins; same basic core structure is conserved between (2)
- catalyzes RNA synthesis
- 3 domains of life
(1) polymerase is the smallest polymerase, with 5 subunits: (2)
- bacrterial
- 2 alpha, 1 beta, 1 beta’, 1 omega
the () subunits of bacterial RNA polymerase form a jaw-like structure
beta and beta’
the () subunit of bacterial RNA polymerase has an N-terminal domain and C-terminal domain joined by a flexible linker
alpha
additional function of RNA Pol II in eukaryotic and archaeal transcription
couples transcription to the processing of the RNA transcript
the () of Poll II is crucial to the coupling of transcription to RNA transcript processing
C-terminal domain (CTD)
core bacterial RNA polymerase requires an extra subunit called () directly contacts (and directs enzyme to) the promoter
sigma factor
in bacterial RNA polymerase, core enzyme + sigma factor = ()
holoenzyme
type of bacterial sigma factor that recognizes promoter sequence for housekeeping genes as well as promote their transcription
primary sigma factor
type of bacterial sigma factor that recognizes promoters for genes whose expression is regulated in response to specific signals or stress conditions
alternative sigma factor
2 elements of bacterial promoters
-35 and -10 elements
some bacterial promoters have extra recognition sequences, like ()
AT-rich UP element
some bacterial promoters have sub-optimal (shorter or nonexistent) -35 elements, which results in ()
extended -10 element
factors that promote sigma factor’s specificity -> regulates bacterial transcription
each sigma factor has preferred binding sequence and preferred spacing element between -35 and -10 elements
in the RNA polymerase holoenzyme, there are 3 sigma domains that are positioned to recognize specific promoter elements
- Domain 2
- Domain 3
- Domain 4
RNA polymerase holoenzyme domain that recognizes the -10 element and performs promoter melting
Domain 2
separation of duplex DNA performed by Domain 2 of the RNA polymerase holoenzyme
promoter melting
RNA polymerase holoenzyme domain that recognizes the 2 bases of the extended -10 element
Domain 3
RNA polymerase holoenzyme domain that recognizes the -35 element
Domain 4
Domain 4 of the bacterial holoenzyme is attached to the flexible part of the core enzyme, which allows it to ()
accommodate different -35 to -10 spacings
some bacterial sigma factors are regulated in response to () conditions
environmental or developmental
bacterial sigma factors can be regulated at the transcriptional or translational level by altering their ()
mRNA or protein stability
one way to regulate sigma factors is by the use of (), which have inhibitory domains that must be cleaved before the sigma factor can associate with the core enzyme
pro-sigma factors
proteins that bind to sigma factors and inhibit their function
anti-sigma factors
in S. typhimurium, the sigma factor () is needed for the expression of genes late in the assembly of the flagellar motility motor
sigma F (alternative sigma factor)
in S. typhimurium, the housekeeping sigma factor () is required for transcription of genes for initial hook and FlgM
sigma 70
in S. typhimurium, anti-sigma factor () binds to sigma F, preventing it from binding to the holoenzyme while proteins that form the flagellum base are being synthesized
FlgM
eukaryotic and archaeal analogs to bacterial sigma factors
general transcription factors
general transcription factor () complex works with RNA Pol II
TFII
TFII proteins assemble at promoters with the core polymerase to form the () -> analogous to bacterial holoenzyme
pre-initiation complex (PIC)
all eukaryotic polymerases need the (1) to initiation transcription (part of 2)
- TATA-binding protein (TBP)
- TFIID
eukaryotic promoters have () that direct binding of pre-initiation complex (PIC) -> analogous to -35 and -10 domain of bacterial promoters
core promoter elements
sequence ~25-20 bp upstream of transcription start in RNA Pol II promoters
TATA box
TFIIB recognition element in RNA Pol II promoters
BRE
initiator element in RNA Pol II promoters
INR
promoter element found downstream of transcription start in RNA Pol II promoters
downstream promoter elements (DPE)
other subunits of TFIID, called (), mediate recognition of other promoter elements like INR and DPE during PIC assembly
TBP-associated factors (TAFs)
an additional large protein complex, called () is needed to activate many Pol II transcribed genes in in vivo transcription
mediator
specific transcription factors for eukaryotic upstream regulatory sequences
activators
Pol I uses other proteins to initiate transcription; it binds to a promoter with a core element recognized by TBP and an ()
upstream control element (UCE)
once RNA polymerase is in position, the RNA-promoter complex is called a ()
closed complex
once transcription bubble is formed, the RNA polymerase bound to an open region of DNA is called the ()
open complex
which RNA polymerases (eukaryotic/archaeal) require ATP
- require ATP: Pol II
- don’t require ATP: Pol I and III (unwinding of DNA is spontaneous)
exception () RNA polymerase requires energy
sigma 54
the non-template strand is held away from template strand by the () regions in RNA Pol
lid, zipper, rudder
similar to DNA replication elongation, () are present at the active site to catalyze addition of ribonucleotides to elongating RNA chain
2 Mg2+ ions (nucleophilic attack mechanism)
(): RNA pol frequently fails to make a full-length RNA on a first attempt -> leads to release of short RNAs (2-9 nucleotides)
abortive initiation
both bacterial sigma factor and eukaryotic TFIIB (involved in abortive initiation) have a () that extends into the RNA pol active site region that must be removed for transcription to continue
loop
process of displacing the loop of sigma factor/TFIIB loop in RNA pol; thought to help the polymerase break away from the promoter
promoter clearance
during promoter clearance, RNA pol undergoes a () that associates it very stably with DNA, and loosens its grip on initiation factors
conformational change
eukaryotic Pol II becomes (1) by the action of (2) as it converts to the elongating complex
- phosphorylated
- TFIIH
once RNA polymerase has transitioned to the (), transcription is highly processive
elongation complex
because the most recently added 9 ribonucleotides are still within the transcription bubble, a () exists within the bubble and associates with Pol to contribute to stabiltiy
RNA-DNA hybrid
RNA polymerase sometimes pauses due to physical obstructions
transcriptional pausing
transcriptional pausing can occur when:
- hairpin forms in RNA transcript (due to short complementary sequences present)
- presence of a weak DNA-RNA hybrid within the bubble (caused by AU-rich sequence or base mispairing)
pausing can be relieved or enhanced by ()
elongation factors
occurs when the polymerase cannot resume RNA synthesis
transcriptional arrest
because the resulting molecule synthesized by RNA Pol II isn’t the final active form, it is called the (), which must be processed
pre-mRNA
the phosphorylated () of RNA Pol II is involved in mRNA processing
CTD region of the Rpb1 subunit
occurs when RNA Pol II is paused at 30-60 bp downstream of +1 and only resumes efficient transcription with the assistance of other proteins
promoter proximal pausing
TFIIH phosphorylates (1) in the (2) as RNA Pol II clears the promoter region -> leads to binding of several negative elongation factors
- fifth serine (Ser-5)
- CTD heptad repeat
addition of guanosine cap to 5’ end of mRNA leads to phosphorylation of (1) in the CTD heptad repeat by (2), which causes the polymerase to resume elongation (after pausing to add the guanosine cap)
- second Serine (Ser-2)
- p-TEFb
the elongation complex can () when there is a pause in RNA synthesis -> allows most recently made RNA to protrude from the front of the complex
backtrack
() chop off the 3’ protruding RNA when the elongation complex backtracks
transcription cleavage factors
transcription cleavage factors chop off 3’ end of mRNA by enhancing ()
endonuclease activity of RNA pol
examples of transcription cleavage factors are (1) in E. coli and (2) in eukaryotes
- GreA and GreB
- TFIIS
histone chaperones include:
FACT, Asf1, and Spt6
2 main classes of bacterial terminators
- intrinsic
- Rho-dependent
bacterial terminators that end transcription in the absence of any other factors
intrinsic (or simple) terminators
2 main features of bacterial intrinsic terminators
- inverted repeated sequence that results in a stem-loop in the RNA
- a string of 8-10 residues that is so unstable it causes Pol to arrest and transcription bubble to collapse
at () terminators, DNA sequence alone is not enough for terminators -> other protein factors are needed
enzymatic
in E. coli, certain genes need the (1) protein to terminate transcription -> called (2); those that do not require the protein are called (3)
- Rho
- Rho-dependent terminators
- Rho-independent terminators
in eukaryotes, intrinsic terminator sites are recognized by (1); these have stretch of (2), which is thought to destabilize the DNA-RNA hybrid in the same way as E. coli intrinsic terminators
- RNA Pol III
- As
Termination for Pol I requires add’l proteins: (1) in yeast and (2) in mice
- Reb1p
- TTF1
termination of Pol II genes is coupled to the processing of the () of the mRNA
3’ end
most eukaryotic mRNAs have a poly-A tail resulting from ()
polyadenylation
2 main models of transcription termination by RNA Pol II
- allosteric
- torpedo
RNA processing proteins associate w processing signals and CTD; cleavage/recognition of the processing proteins causes conformational changes that lead to dissociation of Pol II from DNA
allosteric model of transcription termination by Pol II
after cleavage, RNA downstream of poly(A) side is digested by Rat1 endonuclease (5’ to 3’) -> disrupts polymerization and causes Pol II to dissociate from DNA
torpedo model
in the torpedo model, () acts as the torpedo that degrades nascent RNA until it runs into the RNA pol
Rat1 endonuclease
