Week 1 - Prokaryotic Transcription Flashcards
Recap
- DNA replication
- DNA –> RNA (transcription)
- RNA –> protein (translation)
• RNA replication
RNA –> DNA (reverse transcription)
• majority of RNA in us isn’t mRNA
RNA sequence is
- complementary to template strand
* identical to coding strand
Transcription is
5’ to 3’
on a template that is 3’ to 5’
Coding strand
- nontemplate strand
- the DNA that ha the same sequence as the mRNA
- related by the genetic code to the protein that it represents
RNA polymerase
an enzyme that synthesizes RNA using DNA as a template
• formally described as a DNA-dependent RNA polymerase
Promoter
a region of DNA where RNA polymerase binds to initiate transcription
Startpoint
the position on DNA corresponding to the first base incorporated into RNA (begins at +1)
Terminator
a sequence of DNA that causes RNA polymerase to terminate transcription
Transcription unit
the sequence between sites of initiation and termination by RNA polymerase
• may include more than one gene
Human genome
3 x 10^9 bases but only 1% is exons
• even including introns and exons is only 25%
• only have 22,000 genes
Eukaryote transcription
mRNA modified at 5’ and 3’
• then spliced
• then moves into cytoplasm where translocated by ribosomes
Bacteria don’t contain
introns - so no splicing
Prokaryotic (bacteria) transcription
mRNA is transcribed, translated, and degraded simultaneously in bacteria
• transcription and translation occur at the same time because there’s no nuclear membrane - no barrier
• 0min - transcription begins
– 5’ end is triphosphate
• 0.5min - ribosomes begin translation
• 1.5min - degradation begins at 5’ end
• 2min - RNA polymerase terminates at 3’ end
• 3min - degradation continues, ribosomes complete translation
Eukaryotic transcription
expression of mRNA in animal cells requires transcription, modification, processing, nucleocytoplasmic transport, and translation
• end is polyadenylated
• 25min - mRNA is transported to cytoplasm
• 4hr - ribosome translates mRNA
Transcription occurs by base pairing in a
bubble of unpaired DNA
• RNA polymerase separates the 2 strands of DNA in a transient bubble
• it uses 1 strand as a template to direct synthesis of a complementary sequence of RNA
• the length of the bubble is -12 to 14 bp
• the length of the RNA-DNA hybrid within it is -8 to 9 bp
The length of the bubble is
-12 to 14 bp
The length of the RNA-DNA hybrid within it is
-8 to 9 bp
The DNA is unwound
locally to allow access
• this protects the DNA bases because only opening a amount of double stranded DNA
• stops kilobases of nucleotides from being affected by other chemicals
RNA polymerization
• the 3’-OH group of the last ribonucleotide added to the chain reacts with an incoming ribonucleoside 5’ triphosphate
The transcription has 3 stages
- RNA polymerase binds to a promoter site on DNA to form a CLOSED COMPLEX
- RNA polymerase initiates transcription (INITIATION) after opening the DNA duplex to form a transcription bubble (the OPEN COMPLEX)
- during ELONGATION the transcription bubble moves along DNA
- the RNA chain is extended in the 5’-3’ direction, adding nucleotides to the 3’ end
RNA polymerase binds to a promoter site on DNA to form
a closed complex
RNA polymerase initiates transcription (initiation) after opening the DNA duplex to form a
transcription bubble
• open complex
During elongation the
transcription bubble moves along DNA
• the RNA chain is extended in the 5’-3’ direction,a dding nucleotides to the 3’ end
When transcription terminates/stops
- the DNA duplex forms
* RNA polymerase dissociates at a terminator site
… has the most control over the speed/regulation of the transcription process
initiation
Initiation
- template recognition - RNA polymerase binds to duplex DNA
- DNA is unwound at the promoter
- Very short chains are synthesized and released
Elongation
polymerase synthesizes RNA
Termination
RNA polymerase and RNA are released
Bacterial RNA polymerase consists of multiple subunits
- rpoA
- rpoB
- rpoC
- rpoD
- rpoZ
rpoA
2 α subunits
• enzyme assembly
• promoter recognition
• binds some activators
rpoB
β subunit
• catalytic center
rpoC
β’ subunit
• catalytic center
rpoD
σ subunit
• promoter specificity
rpoZ
ω subunit
• promoter subunit
E. coli RNA polymerase enzyme molecular weight
460 kD
Holoenzyme
the RNA polymerase form that is competent to initiate transcription
• consists of the 5 subunits of the core enzyme and σ factor
Bacterial RNA core polymerases
- 400 kD
* multisubunit complexes with general structure α2ββ’ω
In the DNA bubble the top strand is
separate from the strand being catalyzed but then then new RNA is kept separate
… subunit of RNA polymerase binds DNA
α
RNA polymerase enzyme consists of
- the core enzyme
* σ factor
The holoenzyme can be subdivided into 2 components
- the α2ββ’ω core enzyme that catalyzes transcription
* the sigma σ subinit that’s required only for initiation
Sigma factor changes the DNA-binding properties of RNA polymerase
- the affinity for general DNA is reduced
* the affinity for promoters is increased
The sigma factor gives
RNA polymerase its specificity
How does RNA polymerase find promoter sequences?
- the rate at which RNA polymerase binds to promoters can be too fast to be accounted for by simple diffusion
- RNA polymerase binds to random sites and exchanges them with other sequences until a promoter is found
The rate at which RNA polymerase binds to promoters
can be too fast to be accounted for by simple diffusion
RNA polymerase binds to
random sites and exchanges them with other sequences until a promoter is found
The holoenzyme goes through
transitions in the process of recognizing and escaping from promoters
- When RNA polymerase binds to a promoter
it separates the DNA strands to form a transcription bubble and incorporates nucleotides into RNA
• closed binary complex (RPc) - DNA remains duplex, formation of RPc is reversible
RNA polymerase loses
the sigma factor to move from promoter region
… are recruited for catalysis
Mg ions
• important for catalysis
- Closed complex is converted to
the open complex (RPo)
• by melting a short region of DNA within the sequence bound by the enzyme
• open complex formation is irreversible
• sigma factor plays an essential role in the melting reaction
Formation of RPc
is reversible
closed binary complex
Formation of RPo
is irreversible
Initiation complex contains
sigma and covers 75 bp
- Ternary complex
- the complex in initiation of transcription that consists of RNA polymerase and DNA as well as a dinucleotide that represents the first 2 bases in the RNA product
- there may be a series of abortive initiations before the enzyme moves to the next phase (promoter escape)
- sigma factor is usually released from RNA polymerase when the nascent RNA chain reaches ~10 bases in length
There may be a series of
abortive initiations before the enzyme moves to the next phase
(promoter escape)
Sigma factor is usually released from RNA polymerase when
the nascent RNA chain reaches ~10 bases in length
- General elongation complex
- forms at 15-20 bases and covers 30-40 bp
- promoter escape
- interactions between promoter and RNA polymerase dissolve
(sigma factor is released)
complex covers only 35bp
The sigma factor controls binding to DNA by
recognizing specific sequences in promoters
• a promoter is defined by the presence of short consensus sequences at specific locations
• highly conserved
• the promoter is the region of DNA recognized by the sigma subunit of the RNA polymerase
A promoter is defined by the presence of
short consensus sequences at specific locations
• highly conserved
•
A promoter is
the DNA region recognized by the sigma subunit of the RNA polymerase
The promoter consensus sequences usually consist of
a purine at the startpoint and a hexamer with a sequence close to:
• TATAAT centered at ~-10 (-10 element, Pribnow box, TATA box)
• TTGACA centered at ~-35 (-35 element)
The promoter has 3 components
- -35: TTGACA
- 16-19bp
- -10: TATAAT
- 5-9 bp (startpoint)
the gaps are crucial
The promoter is TA rich
• energetically more favorable to melt the 2 H bonds in TA rather than 3 H bonds between GC
Down mutations
- decrease promoter efficiency
- usually to decrease conformance to the consensus sequences
- Up mutations have the opposite effect
- mutations at the -35 sequence can affect binding or the melting reaction that converts a closed complex to an open complex
- promoter efficiency can be affected by additional elements as well
UP element
a sequence in bacteria adjacent to the promoter, upstream of the -35 element, that enhances transcription
• bound by α subunit of RNA polymerase
• aren’t in every promoter
• used to regulate genes that need to be switched on at high levels all the time
Put picture of promoter thing here
*
Individual promoters usually differ from the
consensus at one or more positions and therefore have different strengths
Different sigma factors recognize
different consensus sequences
• E. coli has several sigma factors
• each causes RNA polymerase to initiate at a set of promoters defined by specific -35 and -10 sequences
• the major E. coli sigma factor is σ70
The major E. coli sigma factor is
σ70
Sigma factors can be exchanged so
attracted to different DNA
Multiple regions in RNA polymerase directly contact
promoter DNA
• the structure of σ70 changes when it associates with core enzyme, allowing its DNA-binding regions to interact with the promoter
The structure of σ70 changes when it associates with
core enzyme
• allows its DNA-binding regions to interact with the promoter
Multiple regions in RNA polymerase directly contact promoter DNA
- multiple regions interact with the promoter
- the α subunit also contributes to promoter recognition
- eg UP elements associated with αCTD
… of sigma determines specificity
the 2.4 helix of sigma determines specificity
The sigma N-terminus controls
DNA-binding
• protein: N-terminal region binds DNA-binding domain in free sigma
• DNA: DNA displaces N-terminus when complex forms
Footprinting is a high resolution method for
characterizing RNA polymerase-promoter and DNA-protein interactions in general
• experiment to find where polymerase binds to DNA by seeing where the gap in the ladder is
Footprinting
a technique for identifying the site on DNA bound by some protein by virtue of the protection of bonds in this region against attack by nucleases
RNA-promoter and DNA-protein interactions in general
• the consensus sequences at -35 and -10 provide most of the contact points for RNA polymerase in the
promoter
• the points of contact lie primarily on one face of the DNA
• asymmetry in interactions
Interactions between sigma factor and core RNA polymerase change during
promoter escape
• a domain in sigma occupies the RNA exit channel and must be displaced to accommodate RNA synthesis
• abortive initiations usually occur before the enzyme forms a true elongation complex
• sigma factor is usually released from RNA polymerase by the time the nascent RNA chain reaches ~10nt in length
A domain in sigma occupies the
RNA exit channel
• must be displaced to accommodate RNA synthesis
A model for enzyme movement is suggested by the crystal structure
- DNA moves through a channel in RNA polymerase and makes a sharp turn at the active site
- changes in the conformations of certain flexible molecules within the enzyme control the entry of nucleotides to the active site
- different channels keep different components separate
Scrunching model of transcriptional elongation
- DNA is pulled into the RNA polymerase holoenzyme as the DNA unwinds to form the open complex
- RNA polymerase holoenzyme unwinds adjacent DNA segments
- the unwound DNA is pulled into the active site during initial transcription
- the unwound DNA re-winds when RNA polymerase holoenzyme leaves the initiation site and moves down the DNA
- energy stored in the system during the scrunching stage is used during promoter escape to break interactions between the holoenzyme and the initiation site to allow RNA polymerase to move forward
so they think the RNA is fixed and instead the DNA moves
Bacterial RNA polymerase terminates at
discrete sites
There are 2 classes of terminators
- intrinsic terminators - those recognized solely by RNA polymerase itself without the requirement for any cellular factors
- rho-dependent terminators - require a cellular protein called rho
Intrinsic termination
requires recognition of a terminator sequence in DNA that codes for a hairpin structure in the RNA product
• the signals for termination lie mostly within sequences already transcribed by RNA polymerase, and thus termination relies on scrutiny of the template and/or the RNA product that the polymerase is transcribing
(almost all sequences required for termination are in transcribed region, hairpin in RNA may be required, RNA polymerase and RNA are released)
Terminators vary widely in their efficiencies
• readthrough
it occurs at transcription or translation when RNA polymerase or the ribosome (respectively) ignores a termination signal because of a mutation of the template or the behavior of an accessory factor
Terminators vary widely in their efficiencies
• antitermination
a mechanism of transcriptional control in which termination is prevented at a specific terminator site, allowing RNA polymerase to read into the genes beyond it
Rho factor
a protein that binds to nascent RNA and tracks along the RNA to interact with RNA polymerase and release it from the elongation complex
rut
rho utilization site
• the sequence of RNA that is recognized by the rho termination factor
Rho factor is a
hexameric ATP-dependent helicase
• each subunit has an RNA-binding domain and an ATP hydrolysis domain
(ENERGY REQUIRED)
• winds RNA from the 3’ end around the exterior of the N-terminal domains
• 5’ end of RNA pushed into the interior
• binding of RNA converts rho into a closed-ring structure
Supercoiling is an important feature o
transcription
• negative supercoiling increases the efficiency of some promoters by assisting the melting reaction
• transcription generates positive supercoils ahead of the enzyme and negative supercoils behind it
• these must be removed by DNA gyrase and DNA topoisomerase
Competition for … can regulate initiation
competition for sigma factors can regulate initiation
• E. coli has 7 sigma factors each of which causes RNA polymeraes to initiate at a set of promoters defined by specific -35 and -10 sequences
The activities of the different sigma factors are
regulated by different mechanisms
• anti-sigma factor - a protein that binds to a sigma factor to inhibit its ability to utilize specific promoters
(indirect mechanism)
• control of transcription at the level of initiation
• it can switch on specific genes if using sigma factor to activate transcription of the gene to make protein
- eg if lots of iron used the iron sigma factor
Sigma factors may be organized into
cascades
A cascade of sigma factors is created when
one sigma factor is required to transcribe the gene coding for the next sigma factor
• the early genes of phage SPO1 are transcribed by host RNA polymerase
• one of the early genes codes for a sigma factor that causes RNA polymerase to transcribe the middle genes
• 2 of the middle genes code for subunits of a sigma factor that causes RNA polymerase to transcribe the late genes
A cascade of sigma factors is created when
1 sigma factor is required to transcribe the gene coding for the next sigma factor
Early genes of phage SPO1
are transcribed by host RNA polymerase
One of the early genes codes for
a sigma factor that causes RNA polymerase to transcribe the middle genes
2 of the middle genes code for
subunits of a sigma factor that causes RNA polymerase to transcribe the late genes
Early genes
phage promoters are recognized by bacterial holoenzyme
• early gene 28 codes for a new sigma factor that displaces bacterial sigma
Middle genes
- gp28 core enzyme transcribes phage middle genes
* middle genes 33 and 34 code for proteins that replace gp28
Late genes
• gp33-gp34 core enzymes transcribe phage late genes
Antitermination can be
a regulatory event
• an antitermination complex allows RNA polymerase to read through terminators
An antitermination complex allows RNA polymerase to
read through terminators
nut
N utilization site
• the sequence of DNA that is recognized by the N antitermination factor
Phage lambda uses antitermination systems for
regulation of both its early and late transcripts
• but the 2 systems work by completely different mechanisms
RNA polymerase holoenzyme that synthesizes bacterial RNA can be separated into 2 components
- core enzyme that is sufficient for elongating the RNA chain
- sigma factor (single subunit) required only at the stage of initiation for regognizing the promoter
Many abcterial promoters have 2 6-pb consensus sequences centered at
• -35
• -10
relative to the start point
The initial closed binary complex is converted to an open binary complex
by sequential melting of a sequence of approximately 14bp that begins in the -10 region and extends to about 3bp downstream from the startpoint
Binary complex is converted to a
ternary complex by incorporation of ribonucleotide precursors
After multiple cycles of abortive initiation, sigma factor
is released
• and the RNA polymerase complex escapes the promoter
• and the core enzyme moves down the template, unwinding the DNA (transcription bubble) as it synthesizes the RNA transcript
The core enzyme can be directed to recognize promoters with
different consensus sequences
by alternative sigma factors
Bacterial RNA polymerase terminates transcription at 2 types of sites
- intrinsic terminators (G-C rich hairpin followed by a U-rich region)
- Rho-dependent terminators (require rho factor, hexameric ATP-dependent helicase)
Transcriptional termination can be prevented by
antitermination complexes
Rho-dependent
• this form of termination can be a form of regulation (RNA polymerase can override it)
• RNA is more stable than DNA
– RNA is single stranded but can form secondary structure using internal hydrogen bonds
– so can fold on itself using complementation hydrogen bonding so can form enzymes (DNA can’t)
• RNA structure important for function - the hairpin formed due to DNA sequence
Rho factor binds to RNA at a rut site and
translocates along RNA until it reaches the RNA-DNA hybrid in RNA polymerase, where it releases the RNA from the DNA
