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Summary 'Microbial Physiology'
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Samenvatting van het vak Microbial Physiology te KU Leuven van prof. Michiels en prof. Steenackers. Bevat afbeeldingen uit de cursus. Behaalde 15/20 in eerste zit.
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Summary Microbial PhysiologyChapter 1: Regulatory systems
Chapter 1.1: Transcription and translation in bacteria
Transcription
o RNA polymerase
Produces all RNAs (rRNA, tRNA, mRNA,..) except Okazaki fragments (primase)
5 sub-units in which σ-factor for initiation of transcription (recycling)
o Transcription initiation
No existing primers required, no helicase required (<-> DNA polymerase)
Specific DNA regions: promoter regions recognized by σ-factor -> positioning
RNA polymerase + unwinding DNA near start site
Rifampicin inhibits initiation by interacting with active site of RNA pol.
o Polymerization
5 promotor elements for binding of RNA polymerase. No promoters with 5
perfect elements because it would result in too strong binding.
Number of RNA polymerases is limited, number of σ is limited -> competition
Formation of a transcription bubble stabilized by 8-9 bp DNA-RNA hybrid
Sigma factor released after initiation -> recycling
Not always continuously: Pause hairpins, backtracks, antiterminators
o Transcription termination: 2 pathways:
Factor independent (intrinsic): GC-rich inverted repeat followed by polyA-
sequence will lead to formation of hairpin loop.
-> formed RNA-RNA complex is more stable than RNA-DNA hybrid + the
multiple interactions between A and U (RNA) are very weak which will lead
to dissociation from RNA polymerase.
Factor dependent: E. coli: 3 transcription terminators: Rho , Tau and NusA
ρ factor:
- Probably universal
- RNA dependent ATPase
- Active on ribosome-free RNA
- RNA-DNA helicase
Mechanism:
- ρ binds to naked RNA, at free rut sites (rho
utilization), only when RNA is not translated
- Moves along RNA (rotates) in 5’-3’ direction
(60nucl/sec) (RNA pol: 100nucl/sec)
- When RNA pol pauses at a Rho-dependent
termination site, RNA-DNA hybrid is separated
- RNA polymerase dissociates and transcription is
terminated
-> Termination of transcription when translation is
terminated
, o rRNA’ and tRNA’s
important role in protein synthesis
Molecular phylogeny: Highly conserved, no horizontal transfer, used for
identifying different species in the gut.
o tmRNA and trans-translation
Double role (tRNA and mRNA)
Ribosome functioning inhibited in the absence of stop codon (release factor
cannot bind)
Encodes C-terminal tag (approximately 10 aa) recognized by Clp protease
Biotech application: destabilise proteins
o Activity of RNA polymerase
Promoter sequences
Sigma factors
Small ligands:
e.g. Guanosine 3’,5’ diphosphate (ppGpp) -> Destabilizes open complexes
Transcription factors
Chromosome structure: supercoiling and interaction with proteins
Translation
o Translation initiation
Translation initiation regions (TIRs):Important for in-frame translation mRNA
- 5’ untranslated region
- Shine-Dalgarno (S-D) – sequence
- Initiation codons
After protein synthesis, formyl groups and N-terminal methionine are
removed by peptide deformylase (A) or methionine aminopeptidase (B).
o Polycistronic mRNA
A single mRNA encodes multiple proteins
Simultaneous translation requires multiple TIR’s, multiple stop-codons
Translational coupling: TIR of second gene forms hairpin loop, initiation
codon is unrecognizable for ribosome unless translation of the first gene
disrupts the secondary structure of the second gene.
Polar effect on gene expression:Mutation in gene1 has polar effect on gene2
- Insertion of transposon (transcriptional terminators in Tn)
- Insertion Ab-R cassette with transcriptional terminator
- Nonsense mutation (deletion, insertion) upstream of translationally
coupled polypeptide
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