Module 6: Reg. of Gene Expression (Post-Initiation Reg.) Flashcards
At what point POST-TRANSCRIPTION can protein synthesis be controlled?
1) Inhibition of transcription AFTER initiation
2) Enzymatic degradation of mRNA
3) Inhibition of mRNA translation
4) Post-translational modification of proteins
What are two main regulatory mechanisms of post-transcriptional regulation?
1) Regulatory RNAs
2) Secondary mRNA structure
–> Including Riboswitches
Aside from mRNA coding DNA regions, all genomes contain regions of DNA…
Regions of DNA that encode for NON-TRANSLATED RNA
Non-Translated RNA
RNAs that do not directly lead to protein synthesis!
–> Do not undergo translation!
What are the types of non-translated RNA?
1) tRNA (transfer RNA)
2) rRNA (ribosomal RNA)
3) sRNA (small noncoding RNA)
sRNA
= “small non-coding RNA”
–> Molecules around 50-400 nucleotides in length that operate by a variety of methods BUT typically are involved in gene regulation by interacting with existing mRNAs
What do sRNAs do generally?
Interact with existing or forming mRNAs causing alterations that impact the mRNA’s completion of transcription or initiation of translation
–> Play a role in gene regulation!
What are sRNAs AKA?
1) miRNA = micro RNA
2) siRNA = Small interfering RNA
3) asRNA = Antisense RNA
Interactions of sRNA with mRNA can…
1) Prevent the completion of transcription
2) Block translation of existing mRNA transcripts
–> Messes with the expression of genes!
What is one example of sRNA in action?
The interaction of an sRNA (dsRA) with an mRNA transcript encoding for sigma-38 (RpoS)!
(Involved when low temperature occurs)
What is wrong with the mRNA produced from the sigma-38 (RpoS) gene?
The mRNA has a hairpin stem-loop secondary structure in which:
1) The ribosomal binding site and start codon are inaccessible
= Ribosome CANT bind and translation cannot occur!
2) RNAse cut sites that if cleaved, break the entire molecule apart
How does sRNA play a role in “fixing” the issues with the sigma-38 (RpoS) mRNA?
A specific sRNA called “DsrA” binds to the RpoS mRNA (along with the Hfq binding protein) to cause a structural change that breaks open the hairpin stem-loop strucutre
== ribosomal binding site becomes active!
AND
RNAse cut site no longer cleaves the entire molecule but just the non-coding leader end!
What sRNA molecule is involved with the sigma-38 (RpoS) mRNA during LOW temperature?
DsrA
What sRNA molecule is involved with the sigma-38 (RpoS) mRNA during cell surface stress?
RprA
DsrA
an 85 nucleotide asRNA molecule that is produced and accumulates within the cell when low temperature conditions occur
–> binds to leader sequence of RpoS mRNA
When does DsrA accumulate in the cell?
During very LOW temperature conditions!
What does DsrA control within the rpoS gene regulation?
Controls whether or not translation occurs!
DsrA accumulated = can bind to rpoS mRNA and translation occurs
DsrA NOT accumulated = doesn’t bind to rpoS mRNA, no translation can occur, and mRNA transcript is degraded!
DsrA present in cell: Process
DsrA present:
1) RpoS mRNA is produced
2) DsrA + Hfq protein bind to the RpoS mRNA
3) Binding triggers secondary structural change of the mRNA = ribosomal site becomes accessible
4) Ribosome binds and begins to translate the RpoS mRNA!!!
== RpoS (sigma factor) is produced; goes to bind to RNA polymerase to direct it to the regulon involved in cold response!
DsrA Absent in cell: Process
DsrA Absent:
1) RpoS mRNA is produced
2) NO accumulation of DsrA = no binding to the mRNA
3) Without DsrA binding, the mRNA CANT be translated
4) mRNA gets cleaved by RNAses and degrades!
=== no translation of rpoS leading to no production of RpoS leading to no activation of the regulon involved in cold response!
Hfq
RNA binding protein that promotes binding of sRNA to mRNA!
In RpoS mRNA, what is causing the formation of the hairpin stem-loop?
The LEADER SEQUENCE of the mRNA
== basepairs with downstream region of the RNA molecule to form the inhibitory secondary structure
What does DsrA need in order to bind to mRNA leader seqeuence?
Hfq!
RprA
A 105 nucleotide asRNA that functions the same as DsrA BUT accumulates under cell surface stress (rather than low temperature)
Leader Sequence
A non-coding region UPSTREAM of the ribosomal binding site (5’ end)
Different sRNAs accumulate in the cell…
under DIFFERENT conditions and stresses
What determines the differential accumulation of sRNAs in a cell?
The different conditions and stresses currently present!
In the case of RpoS production, what is the impact of DsrA?
Presence of DsrA enhances and facilitates translation of RpoS mRNA!
== Allows translation to occur causing the production of RpoS protein (sigma factor!)
HOW do DsrA and RsrA interact with mRNA?
By complementary base pairing to the leader sequence of the mRNA!
How does the RNAse cleavage site on RpoS mRNA CHANGE upon DsrA binding?
The RNAse cleavage site changes from a double stranded region of the mRNA to a single stranded portion of the leader sequence
== Cleavage removes leader sequence!
–> Without DsrA, the cleavage site is positioned at a double stranded region leading to the breaking of the entire mRNA molecule!
Entire DsrA pathway with sigma-38:
What effect can regulatory RNAs have on translation?
1) Can enhance translation (like in sigma-28 and DsrA example)
2) Can inhibit translation
Attenuation
Regulatory mechanism that occurs AFTER initiation of transcription but BEFORE completion of transcription
–> Usually causes EARLY TERMINATION of transcription!
In the trp operon, what is the main DNA “source” of attenuation?
The trpL gene!
Where is the trpL gene located?
In the trp operon, it is the FIRST gene!
== Right following the trp operator –> Is the first gene of the operon to be transcribed!
What does trpL encode for?
Encodes the mRNA leader sequence!
In bacteria and archaea, translation and transcription are…
COUPLED
== they can occur simultaneously!
As a transcript is being formed, the 5’ end of the mRNA can begin getting translated!
What component/s of the trpL gene are responsible for trp operon attenutation?
TWO trp codons at the 5’ end (“beginning”) of the trpL gene!
What are the domains/regions of the trpL gene?
Which domain has the 2 trp codons
4 domains:
Region 1 = HAS THE TRP CODONS (encodes leader peptide)
Region 2
Region 3, Region 4 = Make up the attenuator sequence
trpL gene sequence
From 5’ end:
1) Start codon for leader peptide
2) Leader peptide sequence with 2 trp codons
3) STOP codon for leader peptide
…
4) Region 2
5) Region 3, Region 4 (Uracil rich attenuator sequence)
What is an important feature of the trpL gene that make trp attenuation possible (OTHER than trp codons)?
Regions of internal sequence compatibility
What is the importance of the 4 domains/regions of trpL gene?
They represent regions of the DNA that exhibit internal sequence compatibility
== When transcribed, have the potential to do intramolecular base pairing!
In the trpL mRNA, what regions are able to base pair to each other?
Region 3 can base pair with regions 2 OR 4:
Region 3— Region 2 paired with unpaird Region 4
Region 3—-Region 4 paired with unparied Region 2
3-4 Stem Loop
“Activates” attenuator sequence through the formation of a TERMINATOR LOOP
==3-4 stem-loop terminates transcription!
2-3 Stem Loop
DOES NOT allow for the activation of the attenuator sequence –> Teminator loop never forms
==2-3 stem-loop allows transcription to continue to completion!
What determines whether a 3-4 or 2-3 stem-loop structure forms?
The presence or absence of trp during the translation of the trpL leader sequence!
Translation of trpL: TRP PRESENT
1) trp is present = ribosome “shoots through” translation of the leader sequence and stalls once it reaches the STOP codon!
2) stalling at the stop codon makes region 2 inaccessible for intramolecular base pairing with region 3
3) Region 3 base pairs with region 4 and TERMINATOR LOOP FORMS
4) RNA polymerase dissociates and transcription ends early! (trp operon is not transcribed, stopping trp synthesis!)
Translation of trpL: TRP ABSENT
1) trp is absent = ribosome cannot fully translate the leader sequence as it STALLS at the two trp codons
(never makes it to the trpL stop codon!)
2) Due to stalling earlier in the mRNA, region 2 is ACCESSIBLE for base pairing
3) Region 2 base pairs to region 3 == NO terminator loop!
4) RNA polymerase remains bound and transcription of the trp operon continues to completion
5) Translation begins at start codons of trp operon strucutral genes
== trp synthesis occurs!
trp operon attenuation:
trp synthesis under High trp vs Low trp conditions
High trp = Transcription of operon STOPS early –> No trp synthesis
Low trp = Transcription of operon completes –> trp synthesis occurs!
Under what conditions is the trpL leader sequence fully and NOT fully translated?
High trp = leader seq. FULLY translated
Low trp = leader seq. NOT fully translated
The trp operon is an example of transcriptional regulation via…
STALLED RIBOSOMES and SECONDARY mRNA STRUCTURE
Riboswitches
Sequences of mRNA that bind effector molecules, regulating the completion of transcription or translation of the mRNA molecule
In riboswitches:
The binding and release of effector molecules can trigger…
Changes in the SECONDARY STRUCTURE at the 5’ end of mRNA!
== can inhibit or promote the transcription or translation of an mRNA
What is the difference between transcriptional and translation control of riboswitches?
Transcriptional Control = Involves the formation or removal of TERMINATOR LOOPS
Translational Control = Involves making ribosomal binding site and start codon ACCESSIBLE or INACCESSIBLE
Riboswitch Example:
Repressor molecule binds to a riboswitch of mRNA being actively transcribed
1) Repressor binds to riboswitch
2) Conformational change of mRNA occurs
3) Terminator loop forms
4) RNA polymerase dissociates and transcription STOPS
Riboswitch Example:
Repressor molecules binds to a riboswitch of fully formed mRNA
1) Repressor binds to riboswitch
2) Conformational change of mRNA occurs
3) Inhibitory secondary structure forms = shine-dalgarno seq and start codon become obstructed
4) Ribosome can’t bind to the mRNA = no translation
What types of effectors can riboswitches bind?
Activators and repressors!
Riboswitches are an __________ NOT ____________
Riboswitches are an mRNA equence and are NOT the proteins that bind to the mRNA!
