Microbial Genetics Flashcards
The Central Dogma
-The process in which DNA is replicated, then transcript to mRNA, and then translated to a protein.
Synthesis of the three types of informational molecules
-Replication
-Transcription
-Translation
Replication
-both strands are templates for new DNA synthesis
-DNA strands split and make new one
Translation
-messenger RNA is template for protein synthesis (happens in ribosome)
-look in notebook
Transcription
-one strand is a template for RNA synthesis
-Look in notebook
DNA Structure Bacterial
-circular
-prokaryote
-supercoiled
-plasmids
DNA Structure Archaeal
-circular
-prokaryote
-supercoiled
-plasmids
DNA Structure Eukaryotic
-Linear
-eukaryote
-supercoiled
-plasmids (very rare)
Bacterial Chromosome Supercoiling
-relaxed circular DNA (double strand) then breaks one strand
-Now there is a relaxed nick circular DNA that then rotates one end of the broken strand around helix and seal.
-It is now supercoiled circular DNA
-chromosomal DNA with supercoiled domains
-Look at notes!
What is supercoiling use for?
-to store a large amount of DNA in a tiny space
-When DNA is supercoiled translations and transcription cannot occur.
What is DNA replicarion?
-Semiconservative
-Look at notes
DNA Polymerases
-Pol III
-DNA Replication is carried out by polymerases
-ALL DNA polymerases require an RNA primer to initiate synthesis
-DNA polymerases are involved in a variety of mechanisms used to repair damaged DNA.
-Make copies of chromosomes
-Look at notes
Why do DNA polymerases have proofreading ability?
-to insure fidelity of DNA replication
What things are involved in DNA replication?
-Helicase (enzyme)
-Primase
-Single-strand binding protein
-DNA poly III
-Okazaki fragments
-Look at notes
What does a helicase do?
-splits DNA in half
-unbinding it
What are the two strands that come after the helicase splits the DNA?
-Leading strand
-Lagging strand
What way does poly I work?
-only works from 5’ to 3’
What is the lagging strand?
-this strand is synthesized occurs is a discontinuous manner creating Okazaki fragments
-It does this because pol I can only work a certain way and because of the replication fork
-look at notes
What happens during DNA Replication?
- Dna A protein recognizes the origin and recruits other proteins
-DNA synthesis is ALWAYS 5’ to 3’
-creating lagging and leading strands
-look at notes
What fuses Okazaki Fragments together?
-ligase
Where does DNA synthesis start at?
-origin of replication
what type of origin do prokaryotes have?
-single origin on the single circular chromosome
what type of origin do eukaryotes have?
-many origins on each linear chromosome
What form is replication in?
-“theta-form”
-usually bidirectional
Replication Fork
-where the two strands of DNA are separated and the new strands of DNA are synthesized.
Process of Replication
- replisome binds and initiates synthesis
- replication fork continuous synthesis in opposite direction
- replication forks hit terminus of replication and collide, releasing two chromosome copies
-Look at notes!
What does the DNA gyrase do?
-uncoil DNA
Why does replication go in different directions?
-because it is faster and takes half the amount of time to make a chromosome.
What happens to Okazaki fragments?
-RNA primers start to fill in the gaps between Okazaki Fragments.
-Exonuclease to remove RNA
-DNA polymerase to fill in behind
-DNA ligase
-look at notes
What does DNA ligase do?
-seals the nick!
How are Okazaki Fragments fussed together?
-RNA primer on one end of DNA and DNA poly III on the other
-poly III makes DNA and then poly I replaces it and primer goes away
-poly I is then replaced by DNA ligase and fusses fragments together
-look at notes
What happens during transcription?
-RNA polymerase and sigma recognizes promoter and initiation site.
-Transcription begins, sigma is released and RNA chain grows
-the RNA polymerase separates the DNA strands
-termination site reached, chain growth stops
-polymerase and RNA released
-look at notes
What are operons?
-transcription units
-group of genes located next to each other on DNA
-three main parts: structural genes, operator region, and the promoter
-look at notes
What are operons used for?
-efficient way to expression multiple genes at once
What do operons transcript to?
-operons transcript to polycistronic mRNA
-ORF = open reading frame
What does 1 gene equal?
-mono-cistronic
What type of operon does E. Coli have?
-histidine biosynthesis operon
RNA Polymerase (RNAP)
-Bacterial RNAP
-Eukaryotic RNAP
-promoters contain sequences that are recognized by sigma factors
-have bases from -10 to -35 sequences
Bacterial RNAP
-one core enzyme (4 essential subunits)
-PLUS a SIGMA subunit
-Fast
Eukaryotic RNAP
-many subunits plus other required transcription factors proteins
-Slow
Sigma Factors
-different sigma factors recognize different promoters
-Ex. low nitrogen, so need 30 genes to turn on, call on sigma factors to activate all the genes, sigma 54
Sigma 70
-For most genes, major sigma factor for normal growth.
-TTGACA
Translation - Eukaryotes and Archaea
-have DNA with B recognition (BRE), TATA, and transcription initiation site (Init) that make up the promoter
-it then binds to TATA binding protein (TBP) and Transcription factor B (TFB), start of transcription
-then binding of RNA polymerase
-transcription, look at notes
What two do transcription the same and who does it differently?
-Eukaryotes and Archaea do it the same
-Bacteria do it differently
Gene Structure of Bacterial
-Operons
-y promoter
- (-10) and (-35) sequence bases
Gene Structure of Archaeal
-operons
-promoters
Gene Structure of Eukaryotic
-No operons
-promoters = TATA
What does an Operon in the DNA produce and what two do this?
-produces a polycistronic mRNA
-bacteria and archaea
-look at notes
What do single genes transcribed alone produce and who does this?
-produces monocistronic mRNAs
-Eukaryotes
-look at notes
Polycistronic
-produces multiple protein products
Monocistronic
-contain the coding sequence for only one protein
Transcription: Prokaryotes
-very little non-coding DNA
-virtually no mRNA processing
-Transcription and translation are coupled
-look at notes
Transcription: Eukaryotes
-Have lots of noncoding DNA
-Produce only monocistronic mRNA
-Extensively process mRNA (5’ Cap, introns removed, exons spliced together, 3’ poly A tail)
-Transcription is in the nucleus and translation is in the cytoplasm
-Not coupled
-look at notes
Between introns and exons which ones are coding and which ones are not coding?
-introns are non-coding
-Extron’s are coding
Transcription of Eukaryotic
-DNA with exon and introns transcript
-then primary RNA transcript, RNA processing, cap and tail added and introns excised
-mature mRNA with cap and tail formed
-move to cytoplasm
Translation of Eukaryotes
-Mature mRNA transported to cytoplasm
-has a poly A tail
-translated to form a protein
Translation (mRNA -> protein)
-highly conserved across prokaryotes and eukaryotes
-utilizes an (almost) Universal, triplet, genetic code (three units TTA)
-takes place within ribosomes (three rRNA molecules plus >40 protein)
Ribosomes
-30S + 50S subunits = 70S in prokaryotes
-40S + 60S subunits = 80S in eukaryotes
-small and large subunits of ribosomes (look at notes)
-S = Svedberg Unit
rNA
-binds to mRNA and
-creates peptide bonds
tRNA
-serves as “decoder” between mRNA and amino acid
-codons in mRNA are decoded by anticodons in tRNA
What do antibiotics target?
-The ribosomes
Why does a tRNA exists?
-to read each codon and
-each tRNA is “charged” (covalently linked) to the proper amino acid encoded by that codon.
-look at notes
What is a polysome?
-a complex of multiple ribosomes bound to a single mRNA molecule during translation.
-look at notes
In Bacteria and Archaea, ribosomes bind to…
-sequences in mRNA called…
-“Shine-Dalgarno sequences” or “Ribosome-Binding Sites” (RBS)
-these sequences resemble AGGAGG (core)
-variation can determine strength of translation
Where does translation start?
-at start codon (AUG or GUG) just downstream of RBS in mRNA
What are the first amino acids in bacteria, archaea, and eukaryotes
-N-formyl methionine in Bacteria
-methionine in Archaea and Eukaryotes
What does translation continue through?
-hundreds of codons, adding one amino acid to the new protein at each codon.
Whan is translation terminated?
-at the stop codon sequences
-UAA, UAG, UGA in mRNA
-no tRNA
Translation: Bacterial and Archaea
-start at RBS on mRNA and stop at UAA
-make protein product
Can ribosome binding sites be strong or weak?
-They can be both.
-it effects the strength of translation
Translation: Eukaryotic
-starts at cap on mRNA and ends at UAA
-cap tells where to start and is a modification
-makes a protein product
Translation things for Bacterial
-RBS
-No mRNA processing
-N-formyl methionine
Translation things for Archaeal
-RBS
-methionine
-No mRNA processing
Translation things for Eukaryotic
-Cap AUG
-methionine
-mRNA processing
Which do coupled transcription and translation and who do not and why?
-Bacterial and archaea do coupled transcription and translation.
-Eukaryotic does not because it is more complex
-prokaryotes do because of growth rates
Protein Folding
-the newly translated protein must fold into the proper secondary and tertiary structure in order to be functional.
-The information required for proper folding is built into the primary amino acid sequence for most proteins.
Can proteins fold a lone and can properly folded proteins unfold?
-most proteins can fold a lone, but some cannot
-properly folded proteins become unfolded either spontaneously or due to heat/chemicals.
What happens to misfolded proteins?
-can be aided in proper folding by “Chaperons” such as Dna K/J and GroEL/ES (this requires atp)
-look at notes
Protein Export
-some proteins must exit the cell into the medium or periplasm
-These proteins have a hydrophobic “signal sequence” that inserts in the membrane.
-They are exported in unfolded form via the “Sec system” and fold on the outside.
-look at notes
What system can export a certain fully-folded proteins?
-The Twin-Arginine System (TAT system)
-look at notes
