Microbial Genetics Flashcards
What is Genetics? What does this subject comprise of?
Genetics: the science of heredity
-includes the study of genes
-what genes are
-How they carry information
-How genes replicate and are passed to subsequent generations of cells or other organisms
-How gene expression determines an organisms characteristics
-regulation of gene expression
-gene mutation and prepare
-DNA recombination
What is a gene?
Gene: a segment of DNA or RNA that encodes for a polypeptide or RNA chain that has a function in the organism
Which organisms have RNA genes?
some Bacteriophages and Viruses
Name 6 RNA products that are expressed in either prokaryotic or eukaryotic microbes and describe their function
REVIEW
RNA Products:
1. Ribosomal RNA (rRNA); forms part of ribosomes and catalyzes protein synthesis in ribosome ; translates mRNA into protein
2. Transfer RNA (tRNA); aka as adaptor molecule that is link between mRNA and amino acids sequence. They carry amino acids to ribosome based on mRNA nucleotide sequence (try matching codon with amino acid)
-translate genetic code into proteins
3. SnRNAs (small nuclear RNAs; associate with proteins into complex called Snrnps (small nuclear ribonucleotide proteins; form splicesome complex) and involved in SPLICING.
4. SnoRNAS Small Nucleolar mRNAs); proteins that associate with Nucleolus: they are Guide RNAs to modify Ribosomal RNAs (rRNAs).
5. MiRNAs (microRNAS; regulatory function
6. SRNAs (small RNAs; regulatory RNAs )
Expressed only in eukaryotic: miRNAs, snRNAs
Expressed only in Prokaryotic: SnoRNAs,
Both: rRNAs and tRNAs, sRNAs
(SIDE NOTE: mRNA is NOT Final product, since it will be converted to protein (it is an intermediate)
What are riboszymes?
Ribozymes: RNAS that possess catalytic activity
What is a genome?
Genome: ALL the genetic information in a cell
What is genomics?
Genomics: The study of Genomes -Sequencing and study of all the nucleotide sequences including structural genes, regulatory sequences and noncoding DNA segments
What is a chromosome?
Chromosome: the structure that carries ESSENTIAL hereditary information
Does Bacterial chromosome= bacterial genome?
REVIEW THIS
No bacterial chromosome does not equal bacterial genome, because bacteria can have chromosome and plasmids
bacterial genome: chromsome + plasmid)
What does genome refer to?
Genome refers to the total DNA and RNA of organism.
What are the components of the bacterial genome and what are its features?
Bacterial Genome:
A. Chromosome
1. Most species have ONE CIRCULAR double-stranded DNA chromosome
2. Sizes range from 500-6000 kb
( humans—3.3 million kb- 600 fold increase than bacteria) (5,000 genes vs 21,000)
-hence humans have a lot of DNA and large genes (while bacteria is compacted; not a lot of space)
3. bacterial genome is Looped, folded and attached at one or several points on plasma membrane within nucleoid (folded by supercoiling by DNA gyrase)
4. Carries all Essential genes of the organism.
B. Plasmids
1. Autonomously replicating extrachromosomal DNA
2. Sizes range from 1-300 Kb (1-5% of the genome)
3. **Contain genes that are conditional. NOT necessary for day to day survival but required under certain conditions **
Ex. Resistance, virulence, metabolic pathways (degrading and synthesizing) , bacteriocins, and plasmid transfer genes
-Also carry transposons
What percentage of cell volume makes up E. coli chromosomal DNA? How long is E.coli DNA stretched out.
E. coli chromosomal DNA is COMPACTED to 10% of the cell’s volume
(if you stretched E.coli chromosomal DNA it would be 1 mm long (2um) This 1 mm long DNA is compacted to 10% cell volume.
How can Plasmids be classified? What are the different types of plasmids?
Plasmids can be classified by FUNCTION
types of plasmids
1. Conjugative Plasmid
2. Dissimilation Plasmids
3. R factors/plasmids
(also have virulence plasmids and bacteriocin plasmid)
What is a conjugative plasmid?
Conjugative plasmid: carries genes necessary for Transfer of plasmid (by conjugation) to another cell
What is Dissimilation plasmids? What are examples?
Dissimilation Plasmids: Encode enzymes for catabolism of Unusual compounds
-prevalent in some species;
Pseudomonas known to degrade unusual compounds like (Toluene, camphor, and petroleum hydrocarbons)
-Ti plasmids that encode genes to catabolize opines
TI plasmids: (infect plants cells, and transfer potion of plasmid into plant, trigger plants to produce opines and have enzymes that degrade opines for energy )
WWhat are R factors/Plasmids ?
REVIEW
R factors/Plasmids: Encode resistance to antibiotics, heavy metals, bacteriophages and often….
-carry multiple resistance genes (r-determinants)
-are Conjugative (resistance transfer factor, RTF)
(allows them to transfer resistance gene to another organism)
What Is an example of a conjugative plasmid and what are its features?
Resistant plasmid R100 (a conjugative plasmid)
- Resistance Transfer Factor (RTF): all the genes necessary for transfer
R- determinants: all resistance genes
has resistance for mercury (heavy metal), sulfonamide, streptomycin, chloram-phenicol)
Other structures in plasmid:
(had origin of replication, origin of transfer, Plus and conjugation proteins)
What kind of other unique structures does R100 have?
Plasmid R100 has insertion elements and transposons
What are other functional plasmid groups and what are their structures and functions? How are bacteriocins namd? Provide examples
Other functional Plasmid groups:
1. Virulence plasmids: encode for proteins that enhance pathogenicity of the bacteria
- they allow attachment to host cells
- express toxins (exfoliative toxin (S.aureus) and neurotoxins (C. tetani)
2. Bacteriocin plasmids: encode for bacteriocins (ribosome synthesized peptides that kill closely related bacterial species or even different strains off the same species
- SOME are named after the species its produced by adding “cin” to the species or genus of the bacteria
(ex: Staphylococcus epidermis produces staphylococcin)
(Escherichia coli produces** colicin)
(**Lactococcus lactic produces NISIN) (unique naming)
What is the difference between bacteriocins and antibiotics?
Bacteriocins (polypeptides) are produced by ribosomes and most of them inhibit closely related organisms.
What is a Genotype?
genotype: The genetic makeup of organism/ an organism’s collection of genes
What are the basics of bacterial and Archaea genetic Nomenclature?
Basics of bacterial and Archaea Genetic Nomenclature:
All bacteria and Archaea use the same rules
-Genes are assigned a 3-letter designation (pathway, cell structure, function or mutant phenotype)
-written in lower case and italicized
-Different genes affecting the same pathway are distinguished by Capital letters (ex: fadA, fadB, fadD; 3 enzymes in pathway to degrade fatty acids)
-Each different mutant is assigned a unique allele number (ex: lacZ19)
-
What is an example of E.coli genotype? How do you know what genotype is about?
E.coli genotype
most people name strains based on initials or order generate organism. If you known about physiology of organism, can make guesses of what it means.
MC1061 (name of strain) , araD139 (arabinose D); degradation of arabinose
Delta (araA-leu)7696
galE15 (galactose)
galK16
lacZ74 (lac operon)
rpsL50 (ribosomal protein
hsdR2 (host species defense; function)
argF2 (gene involved in biosynthesis pathway)
**When you write genotype, write names of mutants (defective genes) **
-all mutant alleles have numbers at the end.
Genes are Not listed in the genotype are assumed wild type
other examples:
pilc (synthesis of piling)
lamb (lambadresistant: phenotype)
What is a phenotype ?Describe it more in detail.
Phenotype: the external manifestations of an organism genotype (the observable properties of an organism)
-phenotypes consist of 3-letter symbols, these are NOT italicized, and first letter of symbol is capitalized
when writing symbol for phenotype:
+ indicates a functional phenotype
- indicates Nonfunctional.
Bio- cannot make biotin (but is required as supplement to minimal medium)
,Arg- or Met- ; means it cannot make Arg or Met (but they are required as supplement to minimal medium)
if you have Lac- : you cannot utilize lactose as carbon source
if Gal- : you cannot utilize Galactose as a carbon source
-if there is a r in Str^r it means it is Resistant to antibiotic streptomycin
-if there is a s in Str^s, it means you are Sensitive to antibiotic streptomycin
Describe the components of a Genetic map of the chromosome of E.coli. What are ORF?s What are short tandem repeats and what do they contain? What do the numbers represent in the genetic map?
REVIEW
Genetic Map of the Chromosome of E.coli
1. ORFs: Open reading frame in DNA sequence that lacks termination codons and so can potentially translate as a polypeptide chain
The entire genome does NOT consist of back to back genes (there are DNA between genes that do not code for anything)
2. Short Tandem Repeats (STRs)
-Short DNA sequences (usually 2-5 base pairs) that are repeated numerous times in a head-tail manner.
They are important for use in DNA fingerprinting ( bacterial and humans)
The repeats will be same, but number of repeats will be different.
-The polymorphisms in STRs are due to the different number of copies of the repeat element that can occur in a population
The numbers on the genetic map (0-100) represent Conjugation minutes
(in eukaryotes, introns throw you off in open reading frame; so to help with issue, make CDNA)
Describe the DNA structure and its components
DNA structure
-Polymer of nucleotides: Adenine, thymine, cytosine, and guanine
-Double helix
-“Backbone is deoxyribose-phosphate
-Strands are held together by hydrogen bonds between AT and CG
-Strands are antiparallel
How would you modify the figure to represent dsRNA (double stranded) ?
Get rid of thiamine, replace with Uracil; ADD hydroxyl (OH) group here (to make ribose sugar; 2 OHs)
What is the order of Transcription? What does this mean?
Transcription goes from 5’ to 3’
5’ refers to phosphate and 3’ refers to OH on sugar.
always extend 5’ to 3’
What occurs in semiconservative replication ?
Semiconservative Replication
-1) The double helix of the parental DNA separates as weak hydrogen bonds between the nucleotides on opposite strands break in response to action of replication enzymes
2) Hydrogen bonds form between new complementary nucleotides and each strand of the parental template to form new base pairs
3) Enzymes catalyze the formation of sugar-phosphate bonds between sequential nucleotides on each resulting daughter strand
How does DNA replicate? Explain how.
DNA replicates semi conservativiely.
Strand opens up,
new nucleotides that complement prexisting nucleotides of template come in and form double bonds
enzymes link sugar phosphates together.
before known that DNA replication was semiconservative, it was thought replication was Conserative (1 parent made intact, entirely new double stranded molecule) or Dispersive model
Discuss the differences between semiconservative, conservative and dispersive model. Who were the ones that discovered DNA replication was semiconservative?
Semi conservative model : produces 2 helices in which each helix contains one New strand and One Old (parental ) strand
Conservative Model : produces 2 helices in which one helix contains entirely OLD DNA and other helix contains entirely NEW DNA.
Dispersive model: produces 2 helices in which each strand contains alternating segments of Old and New DNA
-Messelson and Stahl discovered DNA replication was semi-conservative
What is the role of DNA polymerase, DNTPs, and nucleoside in DNA Synthesis?
DNA Synthesis
-DNA Polymerases only add new DNTPs to the 3’ end
-Energy is supplied from phosphates of DNTP’s (deoxynucleoside triphosphate)
-When a nucleotide triphosphate bonds to the sugar, it loses two phosphates.
-Hydrolysis of the phosphate bonds provides the energy for the reaction
Differentiate between nucleoside and nucleotide
nucleoside: Base and sugar
Nucleotide: base, sugar and phosphate (either 1, 2 or 3 phosphates)
What is the process of DNA synthesis and what structures are included ? What is the rate of error for E.coli DNA polymerase? What further reduces error.
DNA Synthesis:
DNA is copied by the DNA polymerase
-in the 5’ –> 3’ direction
-Leading strand is synthesized continuously
-Lagging strand is synthesized discontinuously (Okazaki fragments) and initiated by an RNA primer
-RNA primers are removed by a DNA polymerase and Okazaki fragments joined by DNA ligase
-Overall error rate of E.coli DNA polymerase is low (10^-5) then further reduced to a 10^-9 bases due by PROOFREADING
List out the steps of DNA synthesis ?
Steps of DNA Synthesis :
1) Enzymes unwind the parental double helix
2) Proteins bind stabilize the unwound parental DNA (single stranded binding proteins ; ssbps)
3) The leading strand is synthesized continuously by DNA polymerase
4) The lagging strand is synthesized discontinuously. Primase, an RNA polymerase, synthesizes short RNA primer, which is then extended by DNA polymerase
5) DNA polymerase digests RNA primer and replaces it with DNA polymerase
6) DNA ligase joins the discontinuous fragments of the lagging strand
Discuss the important enzymes in DNA replication and their functions
Important enzymes in DNA Replication, Expression and Repair:
1. DNA Gyrase: relaxes supercoiling ahead of replication fork; (Generates (-) supercoils)
2. **DNA Ligase: makes covalent bonds to JOIN DNA strains; Okazaki fragments and new segments in excision repair
3. **DNA polymerases: Synthesize DNA; proofread and facilitate repair of DNA
4. Endonucleases: Cut DNA backbone in a strand of DNA; facilitate repair and insertions
5. **Helicase: Unwinds double stranded DNA
6. Methlyase: adds methyl group to selected bases in newly made DNA
7. Photlyase: uses visible light energy to separate UV-induced pyrimidine dimers
8. **Primase: An RNA polymerase that makes RNA primers from a DNA template
9. Ribozyme: RNAs that possess catalytic activity (can catalyze chemical reaction)
10. **RNA Polymerase: Copies RNA from a DNA template
11. SnRNP: RNA protein complex that removes introns and splices exons together
12. Topoisomerase or Gyrase: Relaxes superocoild ahead of replication fork; Separates DNA circles at the end of DNA replication
13. Transposase: Cuts DNA backbone, leaving a single-stranded “sticky ends”
How are DNA Gyrase and Topoisomerase similar and different ?
BOTH DNA Gyrase and Topoisomerase RELAX Supercoiling
TYPE I Topoisomerase; that RELAXES supercoiling and cuts ONE DNA strand .
-DNA Gyrase is like a form of type II Topoisomerase that RELAXES supercoiling (NEGATIVE Supercoiling) and REDUCES supercoiling. It cuts TWO strands of DNA
Are any of the enzymes listed above) prokaryotic or eukaryotic specific?
Yes, DNA Gyrase is Prokaryotic specific: only bacteria
snRNPS; (small nuclear ribonucleotides proteins) are only eukaryotic
(the rest of enzymes are found in both)
Describe the E.coli DNA replication process? How many origins of replication and replication forks are there? What separates the two loops in replication?
How long does it take bacterium E. coli chromosome to replicate? Vs Doubling time?
E.coli DNA replication is BIDIRECTIONAL
it has **single origin of replication*
Process:
The replication initiates at origin of replication and Polymerase moves in 2 different directions (bidirectional)
-2 replication forks that will continue until you have gone all around chromosome and separate two loops with Topoisomerase
Then, two genomes get attached to cell membrane at opposite poles, so that when cell divides you segregate chromosomes into different cells
After duplication, each copy of the origin binds to the membrane at OPPOSITE POLES
bacterium E.coli requires 40 minutes to finish replication time for chromosome
-but the doubling time of organism is as fast as 20 minutes
(2 replication forks, one continuous side, other side discontinuous)
Explain how it Is possible for E. coli chromosome replication to be 40 mins but the doubling time to be as fast as 20 minutes.
because when organism initiates replication every 20 minutes; after first initiation starts at origin of replication, it makes a complete genome every 20 minutes.
It initates a new set every 20 mins, so hence completes another chromosome every 20 minutes (1 is halfway done)
What occurs in Bacterial Transcription? When does transcription start and stop?
Bacterial Transcription:
-DNA is transcribed to make RNA(mRNA, tRNA, rRNA, etc)
-Transcription begins when RNA polymerase binds to the PROMOTER sequence
-Transcription proceeds in the 5’ to 3’ direction
-Trancription stops when it reaches a TERMINATOR sequence
(RNA polymerase is bound to DNA)
Describe the process of transcription in Prokaryotes. What are the steps and components involved?
Process of Transcription in Prokaryotes:
1) RNA polymerase binds to the promoter, and DNA unwinds at the beginning of a gene
2) RNA is synthesized by complementary base pairing of free nucleotides with the nucleotides bases on the template strand of DNA
3) The site of synthesis moves along DNA; the DNA that has been transcribed rewinds.
4) Transcription reaches the terminator
5) RNA and RNA polymerase are released the DNA helix re-forms.
Compare and contarast Prokaryotic vs Eukaryotic RNA Polymerases
Prokaryotic RNA polymerases: single polymerase with 1 transcriptional factor (sigma factor that regulates the polymerase)
Eukaryotic RNA polymerases: 3 types of RNA polymerases, and lots of transcription factors
Compare and contrast Bacterial Rho dependent vs Rho Independent Termination
Bacterial Rho dependent Termination : Nucleotide sequences are recognized by Rho protein, which bind sequence and cause polymerase to come off
Rho independent Termination: sequence dependent; NO proteins involved in sequence. When RNA is made, it will form stem loop structure.
Stem is G-C rich (strong stem ) and stem loop is followed by a string of U’s (uracils) causing polymerase to fall off
What occurs in mRNA transcription and processing in Eukaryotes? What are the steps?
What further Eukaryotic mRNA modification are NOT shown?
mRNA Transcription and Processing in Eukaryotes:
1. In the nucleus, a gene composed of exons and introns is transcribed to RNA by RNA polymerase
2. Processing involves snRNPS (small nuclear ribonuclear proteins) in the nucleus to Remove the Intron-derived RNA and Splice together the Exon-derived RNA into mRNA
3) After further modification, the mature mRNA travels to the cytoplasm, where it directs protein synthesis
Further Eukaryotic mRNA modifications Not shown:
-5’ Methyl guanosine Cap at 5’end (beginning of sequence)
-Poly A tail added at end of sequence
Introns are removed in the nucleus before mRNA gets transported out of the cytosol (where translation occurs)
What occurs in translation? When does it start and end?
Translation (process of converting mRNA into protein)
-mRNA is translated into CODONS (three nucleotides)
-Translation of mRNA begins at the START codon: AUG
-Translation Ends at NONSENSE Codons; UAA, UAG, UGA
Describe the genetic code and it’s components. What structure carries the anticodon?
The Genetic Code
-61 sense codons on mRNA encode the 20 amino acids
(total of 64 codons; if include 3 nonsense/stop codons)
-The genetic code is DEGENERATE (several codons can code for same amino acid)
EXCEPT AUG (Methionine) has 1 codon
-tRNA carries the complimentary ANTICODON
(anticodon is the trinucleotide sequence that is complementary to codon in mRNA)
tRNA charged with Amino acid to match mRNA
What is purpose of Degeneracy in Genetic Code? What does the start codon encode for in bacteria?
Degeneracy enables SILENT Mutations (if change the last codon ,you still have same amino acid)
ex; Serine has 4 different codons that code for same aa (can change 2 of nucleotides in codon)
-in bacteria, the start codon encodes for N-formylmethionine
(specific tRNA carries N-formylmethionine )
What is Codon bias?
Codon Bias: The probability that a given codon will be used to code for an amino acid over a different codon ,which codes for the same amino acid.
Do all bacterial proteins in the cell contain N-formylmethioniine was the first amino acid? Explain why or why not
NO, because most of them (start codons: Nformlymethioine) become processed and removed. Although they start with the codon that encodes for N-fomylmethionine, they have enzymes that will remove it
What are the necessary components for the initiation of translation?
Necessary components for initating translation:
- 2 ribosomal subunits (large and small_
-tRNA,
-Anticodon (charged with amino acids)
-Methionine (start codon)
mRNA
How is the correct AUG start codon recognized in prokaryotes vs Eukaryotes?
Recognize correct AUG (start) codon:
in Prokaryotes,
In Eukaryotes, Uses SCANNING Model: Cap (at 5’ end) is recognized by the ribosome and when it binds to the ribosome, it scans it for the first AUG.
( CAP protects 5’ end and is needed for ribosomes to translate )
In prokaryotes, use Shine Dalgarno Sequence. Upstream of correct AUG, a few nucleotides up, there is a short sequence called shine Dalgarno. This sequence is complementary to sequence in ribosomal RNA, which allows Ribosome to recognize where it should start.
(shine dalgarno sequence matches sequence in ribosome, to align it and be translated.)
prokaryotes have Shine dalgarno sequence allow ribosome to align with AUGs, also have operon: 3 different start codons and 3 different stop codons
What is an operon?
Operon: group of genes that are under control of single operator. Multiple genes on a single RNA transcript
(operons are bacteria specific)
(eukaryotes doe NOT have operons; since they cannot internally load)
Describe the steps of translation? When is the only time amino acid is in her favorite o
Translation
1. On the ribosome, when ribosome binds DNA, there are three sites, E site (Exit) P site (peptidyl) and A site (Aminoacyl)
2. When you first initiate translation , the first tRNA goes to the P site (only time get amino acid in P site is at initiaton of translation) (subsequently tRNA goes to A site). So have start codon (formylmethioine in bacteria) and next tRNA
3) Another tRNA goes to A site (carrying Leucine) , peptide bond formed between Methionine and leucine
4) methionine will then be released from tRNA and ribosome will move along mRNA in 5’ to 3’ causing tRNA (that had start codon) to move to exit site and tRNA carrying leucine (attached to Met) to P site.
Also another tRNA carrying with another anticodon (that codes for GGU) another Amino acid (glycine) will enter A site. –Transfer leucine to glycine to form peptide bond
5) 3 tRNAS in the E (Aug start codon), P site (leucine with Methionine attached) and A site (Glycine). This process keeps going (growing polypeptide chain) on until reach end of gene. Then, Nothing enters A site because of STOP codon and polypeptide is released from tRNA.
6) Ribosome comes off and new protein is made.
How are the processes of transcription and translation in prokaryotes?
In Prokaryotes, transcription and translation are COUPLED
(RNA polymerase (catalyzing transcription) and Ribosome (catalyze translation) can bind to same RNA transcript
As soon as polymerase starts making mRNA, polymerase transcribes
as soon As AUG code in shine dalgarno sequence are exposed, they are bound by ribosome and start translating before whole mRNA is made
As soon as Ribosome moves past AUG , and moves farther down transcript and Aug is exposed again another ribosome will bind hence Polycistronic ( mRNA that encodes for multiple different protein products)
Transcription and translation occur at the same time (they are coupled)
Why aren’t these processes (transcription and translation ) coupled in eukaryotes?
Transcription and translation are NOT coupled in Eukaryotes.
This is because in eukaryotes, Transcription occurs in Nucleus; mRNA is processed in multiple different ways
only when mRNA is fully processed, its exported to cytosol for translation .
Hence translation and transcription cannot occur at the same time in eukaryotes.
Describe the genes that are involved in transcriptional control of bacteria. Explain the role of Constitutive, Repressible, and inducible genes. what is Catabolite repression?
Transcriptional control in bacteria
-Constitutive genes (60-80%) are expressed at a FIXED rate (not regulated in any way; expressed at same level)
-Other genes are expressed only as needed
-Repressible genes: transcription rate decreased by a regulator protein called a repressor. Default position is ON
-Catabolite repression: the INHIBITION of synthesis of alternative catabolic enzymes by a preferred carbon source (ex; glucose)
(if you already have glucose, you shut off transcription of other enzymes, since glucose is preferred)
Inducible genes: transcription rate Increases by a regulatory proton called an Inducer. Default position is OFF.
(protein binds promotor and increases transcription rate)
Discuss the negative and positive regulation that occur in the lac operon
Lac Operon has both negative and positive regulation
structure:
*I region is the repressor. It is NOT regulated and has its own promoter
*The operon genes for lac operon: ZYA is expressed from a single promoter and has operator
operator: a sequence that a repressor binds
(control region structural genes: PO ZYA)
-P is promoter
-O is Operator
What occurs in Negative regulation of the lac operon?
Negative regulation of the Lac operon:
The repressor mRNA is produced and converted to protein. That repressor binds the operator sequence and prevent RNA polymerase from moving forward and hence BLOCKING transcription
Repressor Active, Operon OFF
What occurs in the Induction of the lac operon? What are the enzymes used for this process?
Induction of Lac operon:
A small molecule called Allolactose (inducer) binds to repressor, it will cause lac operon to come off
-Allolactose: disaccharide, similar compound to lactose that is generated when you bring lactose into the cell.
(lactose itself does not affect repressor)
beta galactosidase : enzyme that cleaves disaccharide to glucose and galactose. It cleaves a beta bond between the two sugars
Repressor Inactive, Operon still OFF
permease : transport enzyme that brings in lactose
Transacetylase; enzyme that puts acetyl group on lactose
lactose: disaccharide of glucose and galactose
If you have lactose around, you can use enzymes to degrade lactose.
operon is still off, since you need something for RNA polymerase to bind.
Just removing repressor does not turn on operon (need activator)
