Question

Microbiology Tutorial: Basics of Microbial DNA Synthesis, Transcription, Translation

Answer 1

\({\bf{RNA:}}\)
rna - has 2' hydroxyl on ribose molecule while dna only has h group, hence deoxyribose vs ribose
uracil lacks methyl group on carbon 5

\({\bf{Enzymes/Structures:}}\)
- DNA polymerase I: removes RNA primers, high abundances
- DNA polymerase III: adds nucleotides, low abundance, 5 - 3' direction
- RNA primase: makes primer
- RNA polymerase: opens DNA strand for RNA synthesis
- DNA ligase: joins the nucleotides together
- mRNA makes proteins
- rRNA makes up the ribosome
- tRNA links amino acids in translation
- promoter: site where RNA polymerase binds
- transcription start site: (+1), close to promoter, upstream of AUG codon
- sigma factors: bacterial transcription factors, ex. sigma70

Answer 2

\({\bf{Terminating~Transcription:}}\)
- rho independent: uses termination sequence with high GC content and many U residues, GC content causes segment to form into stem loop structure terminating transcription
- rho dependent: rho is a protein that uses ATP to unwind the DNA/RNA hybrid (less common)

- operons: groups of related genes
- polycistronic: multiple genes can be transcribed from one transcript
- in bacteria, transcription/translation occur simultaneously

\({\bf{Basic~Mechanism~of~Translation:}}\)
- aminoacyl-tRNA synthetase: joins tRNA to amino acid at 3' end, capable of proofreading
- anticodon base pairs with codon
- N-formylmethionyl-tRNA: replaces normal methionine in AUG codon, has amino group blocked so it can only be attached to the N-terminus
- Shine Dalgarno sequence: located upstream of the start codon, purine rich, guides initiation of synthesis
- initiation complex: forms after hydrolysis of GTP w/ addition of initiation factors
- A site (aminoacyl), P site (peptidyl), E site (exit)
- binding of tRNA to A-site --> hydrolysis of GTP --> release of gdp-EF-Tu complex, recycling Ef-Tu
- shifting of two amino acids to empty the A-site
- termination codon enters A-site, release factor binds to A site and the linkage is hydrolyzed, releasing the polypeptide

Answer 3

- dnaA: binds to the origin of replication, positive initiator
- dnaB: unwinds DNA, AT rich, binds to site 13 bp into the oric
- single stranded binding protein (SSBP): binds to strand and prevents DNA strands from rejoining

- subunits of RNA polymerase:
alpha - recognizes the promoter
beta - contains the polymerase
beta prie: binds to DNA
omega: renatures polymerase

Answer 4

\({\bf{Plasmids:}}\)
- origin of replication, determines host range
> ColE1: narrow range
> RK2: broad range

- two mechanisms
> rolling circle
> theta

- Rolling Circle:
- plasmid nicked, then 3' end "unrolled" and used as template for replication

- RNA II primer used for DNA plasmid synthesis
- RNA I: transcribed from opposite side
- Rop: prevents cloverleaf, stabilizes hybrid
- RNAse H: processes RNA II for replication

Answer 5

\({\bf{Basics~of~PCR:}}\)
- high T, melt DNA
- cool DNA, primer binds
- increase T again, DNA pol III synthesizes complementary strand
- repeat as needed to increase product

plasmid DNA can be isolated through alkali lysis (high salinity and pH --> denaturation of DNA)

\({\bf{Basics~of~Plasmid~Insertion:}}\)
- choose plasmid compatible with host
- choose restriction enzyme site
- choose primers that incorporate the restriction enzyme sites needed for the particular gene to insert

Answer 6

Anyway that's the end, I hope this is a useful resource. I am not an expert by any means but I will attempt to answer questions to the best of my ability.