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Part II: Studying Gene Regulation in Bacteria
Terms in this set (12)
What is the general structure of the bacterial gene?
- promoter characterised by the -35 and -10 sequence that bind to the sigma factor of a ribosome.
-Operator site that allows effectors to bind and regulate either positively or negatively.
-RBS: ribosome binding site characterised by a shine-dalgarno sequence - complementary to the 3' end of the 16S rRNA
-ORF: open reading frame - nucleic acid sequence that codes for a protein
- RNAs to begin with tend to do nothing and then they form a sequence that conforms to make a ribosome transcription.
Outline what 'Transcriptional reporter fusions' (promoter fusions' are and give examples.
- the promoter that encodes one gene has been fused with the protein coding sequence.
- implies that we know where the +1 is and that may not be very easy.
- could attach the lacZ gene that encodes beta-galactosidase.
What are the advantages and disadvantages of Promoter fusions?
-Best transcriptional reporter: reports 100% of promoter activity.
-No complications due to regulated mRNA degradation (RNase E) or other post-transcriptional regulations.
-Forms a deletion mutant if made by gene replacement
-Difficult to construct : fusion at +1
-Multiple promoters may be present.
What is the 'Transcriptional Reporter Fusions' (Quick-N-Dirty)?
-when the original protein coding sequence is not completely spliced out
- the protein that will be inserted is placed further upstream so there is no chance of a deletion or interrupting the promoter.
What are the advantages and disadvantages of transcriptional reporter fashions?
- easy to construct, no worries with +1 or multiple promoters.
- Never forms a deletion mutant.
- High risk that the stability of upstream mRNA is affected and unaccounted for.
- Reporter production may not reflect the promoter activity
What are 'Transcriptional Reporter Fusions' (Non-Disruptive fusions)?
The reporter protein gene is replaced further down the one we want to study. There are no truncations of either protein and they run one after the other.
What are the advantages and disadvantages of Non-disruptive Fusions?
- Easy to construct.
- No worries with +1 or multiple promoters
- Never forms a deletion mutant.
- High risk that the stability of the upstream mRNA is affected and unaccounted for.
- Reporter production may not reflect at all promoter activity.
What are translational fusions when the promoter matches that of the first gene encoded?
Translational fusion is when the the two protein coding sequences are fused together to form a chimera DNA sequence. both the genes are in their open reading frame so that the correct protein sequence can be formed.
what are the advantages and disadvantages of translational fusion when the promoter match the first protein DNA sequence in the chimera DNA?
- No worries with +1 or multiple promoters.
- Reporter production reflects transcription and translation
- May be difficult to construct (in-frame fusion)
- May form a deletion mutant
- May be subject to artefacts not reflecting actual target gene expression depending on where it is made
Give an example of translational fusion where the promoter and first gene match.
-arcDABC in Pseudomonas aeruginosa arginine deaminase pathway operon
- in the cell the ArcA is produced more the than ArcD which means the promoter is stronger.
-they tried to form a chimera protein with arcD and LacZ but the transcriptional level dropped because the arcA promoter was actually placed in the middle of ArcD and ArcA. they have spliced out the main promoter.
what are the advantages and disadvantages of a translational fusion where the promoter matches the second protein in the chimera protein coding sequence?
- Best translational reporter: reports 100% of translational control
- Not likely to form a deletion mutant - preferably carried on a plasmid.
- Most difficult kind of reporter to construct.
- May be subject to artifacts not reflecting actual target gene expression.
How can you test the post-transcriptional control?
With the addition of either a translational repressor that promotes mRNA degradation via RNases or by translational activators that prevents RNases from binding
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