Lecture 10 Flashcards
Regulating translation initiation:
- Protein binds mRNA regulating initiation at SD/AUG (influencing ribosomal binding)
- sRNA binds mRNA regulating initiation at SD/AUG
- Structure in mRNA inhibits initiation at SD/AUG, could be a small MW effector molecule or a ribosome translating an upstream ORF.
Translation:
- Most ORF have their own SD sequence with the initiation codon at the start
- Each ORF is translated by independent ribosomes
SD sequence:
- Complementary to the 3’ end of the 16s RNA in the ribosome, so it positions the ribosome ready for translation
General mechanisms for regulation translation initiation:
- AN RNA-binding protein prevents translation initiation by blocking the ribosome site
- A secondary structure in the mRNA prevents translation initiation of the second ORF, so translation of the first ORF disrupts the structure allowing translation.
Ribosomal proteins:
- Large subunit
- Small subunit
- Expressed in a series of different operons
Regulation of ribosomal proteins:
- Some ribosomal proteins interact with rRNA forming an integral structure.
- If rRNA is in excess, proteins will be used.
- If ribosomal proteins are in excess, protein 2 binds to mRNA preventing translation of protein 1.
- Translation of ORF2 is inhibited by structure in mRNA
eg) Ribosomal S8 normally binds structure A in the 16S rRNA:
- If in excess, ribosomal protein S8 binds structure B in the mRNA encoding S9 thus inhibiting translation of itself
- Feedback regulation
Riboswitches:
- can regulate translation initiaition and influence transcription termination
eg) SAM is a small MW effector molecule that alters the structure of mRNA to block the ribosome binding site, blocking transcription.
Trans-acting small RNAs (sRNAs):
- Can regulate translation initiation positively or negatively
- sRNA binds to mRNA inhibiting formation of secondary structures, allowing ribosome to initiate at SD/AUG
- or Preventing translation initiation at SD/AUG
Osmolarity response - involve an sRNA
- The regulator allows one gene to be positively regulated while the other gene is effectively negatively regulated.
- small RNA response
OmpF
a protein that forms pores in the membrane
OmpC
A protein that forms pores in the membrane
ompF:
- A porin gene
OmpR:
- positively regulates ompC
- positively regulates the small RNA gene, micF
- Inhibits ompF expression
micF
- A negative regulator of ompF
- A small RNA gene, which binds to ompF mRNA inhibitin translation
Regulation of translation termination eg) Translation release factor 2 (RF2) regulates its own expression:
- Excess RF2: ribosomes terminate RF2 translation at codon 26, and expression doesn’t occur
- Limiting RF2: ribosomes do not terminate RF2 translation, ribosomes shift reading frame, due to an internal SD sequence that repositions the ribosome by 1 base, to translate full ORF
How does density effect bacterial gene expression?
- Cells can uniformly change in some way by activating expression of response genes
- A signal produced reaches a critical threshold which activates a synchronised response
Examples of this:
- Disease causing bacteria expressing disease causing genes only once they reach a certain level of population density
- Biofilms
- The trp operon is regulated by an attenuation mechanism
- Some trpR- mutants are dominant (ie: trpR-D)
- Tryptophan is the effector of the trpR regulatory protein
- Transcription termination involves the formation of a stem-and-loop in the RNA
The trp operator is a region of dyad symmetry. Explain: 2 gives the best reasoning
- TrpR is a dimer, so a mutant monomer can interfere with the function of a WT monomer and these are generally mutations in the DNA binding domain.
- Region of dyad symmetry, but nothing todo with the operator or the binding of TrpR
Unable to undergo lysis or form a lysogen (lethal mutation)
N mutant: because N is needed to progress the intermediate early genes, and if this is mutated it is lethal
Unable to establish a lysogen
cI mutant: because lambda repressor is inactive and PR and PL cannot be repressed
Unable to undergo lysis
Q mutant: unable to expressed late genes
Xis: could undergo lysis, could undergo lysogeny, could integrate, but wouldn’t be able to excise, so it can’t fully enter the lytic cycle
Unable to both undergo lysis OR form a lysogen but lysogen cannot be induced to undergo lytic cycle
nutL mutant: required for cIII expression, more likely to undergo lysis, possibly unable to establish a lysogen
More likely to form a lysogen than a WT lambda
OR3 mutant: overlaps PRM, so it is involved in Cro binding to inhibit cI expression, so more likely to get lysogeny
PRE mutant: repressor establishment, mutation here means it would be similar to a cII mutant, unable to form a lysogen
