Feb 13 Flashcards
(30 cards)
Most genes in prokaryotes are found in _________>
Operons
What are operons?
Where most genes located in prokaryotes, they are clusters of about 3 genes with same promotor, get 1 mRNA transcribed, the genes are related.
What is a promotor, how used directly and indirectly?
It is any DNA sequence that regulates gene transcription. It can have direct RNA pol binding, or indirect interaction with enhancers using transcription factors.
What is at -10 and +1 sites of promotors? WHy have lots of A and T at -10 region?
+1 is start of transcription, -10 region is TATAAT. AT have 2 H bonds, easier to separate strands at -10
Where are 3 locations other promotor elements can be located in eukaryotes?
Upstream, downstream, in intronic sequences (in introns)
What is the 5’UTR in eukaryotes and prokaryotes?
Prokaryotes: Ribosome binding site.
Eukaryotes have 5’ mRNA cap which is guanine methylated on position N7, or internal ribosome entry site (IRES)
What is the name and sequence of translation initiation sequences for eukaryotes and prokaryotes?
Eukaryotes: Kozak sequence GCC-ACC-AUG. The last A is +1
Prokaryotes: The translation initiation sequence is called Shine-Dalgarno, it is AGG-AGG-8 base pairs,-AUG. This last A is +1.
Do all bacterial genes use ATG for start codon? What else do they use? What amino acid do you get for them?
Abput 80% of genes use ATG, others are GTG adn TTG, so just not CTG. Alternative start codons still lead to methionine.
How many possible open reading frames (ORFs) in DNA strand? How many possible in mRNA?
There are 6 possible reading frames in DNA, 3 are forward and 3 are reverse.
mRNA has 3 possible.
How splicing occurs? What is the sequence?
Have GU splice donor, then A at branch point, then AG is splice acceptor.
- The 2’OH of A will attach the PO4 of G at 5’ splice site, releasing exon
- The exposed 3’OH of 5’ exon will then attach PO4 of the G of 3’ splice site, so now join exons and leave the intron behind
What is consititutive splicing?
All introns removed
What are the 3 stop codons?
UAG, UGA, UAA
What are the 2 release factors that bind in A site of ribosome to stop codons that cuase ribosome to release peptide?
eRF1 and 3
How does transcription termination in prokaryotes occur, there are 2 ways
Intrinsic termination: Hairpin disrupts RNA pol, used by expression vectors.
Enzyme-dependent: Translocase disrupts transcription
Does Eukary RNA Pol 2 have transcription termination signal? What is polyadenylation signal and how play role in stopping transcription?
No it doesn’t, just falls off.
It is AATAAA, this recruits polyadenylation and cleave factors, cleave mRNA. Then add Poly A tail.
Do prokaryotes polyadenylate their mRNAs?
No
What are some examples of mechanical lysis of cells? Chemical,
Manual grinding, blender, freeze-thaw, sonication.
Detergents like SDS, put cells in hyptonic solution so cells burst, chaotropic agents like urea so Hphobic MB solubilizes, EDTA that chelates Mg2+ and blocks DNase activity, disrupts cell walls
What are 2 key enzymatic lysis enzymes?
Lysozyme, will cleave peptidoglycan links in gram pos cell wall.
Proteinase K will digest nucleases
Why phenol and chloroform extraction for DNA?
Phenol dnatures proteins, preciptates them. Chloroform will do the same and help separate organic and aqueous phase, DNA and RNA in aq phase
Why ethanol and sodium acetate precipitation for DNA?
It will precipitate nucleotides, used to clean or concentrate DNA
How does Silica filter extraction DNA work?
DNA and RNA binds to silica MB in high salt, EtOH does not elute it. Low salt (water) will elute DNA
What order do proteins, RNA, DNA elute when adding more salt in ion exchange chromatography?
It is in the order of proteins first, then RNA, then DNA as it is the most charged
What is a CTAB extraction?
It is cetyltrimethylammonium bromide. It denatures polysaccharides, precipitates DNA, so good for plant tissues.
What is used to extract RNA?
Use phenol and other stuff, it has 3 layers, results in RNA in aq, and proteins and DNA in organic
