Summary 'Microbial Physiology'

Summary Microbial Physiology
Chapter 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