Studied by 67 people
DNA replication, transcription, translation, Protein Folding and Secretion, Gene Expression Regulation, HGT, and Complex Processes
Define replication.
The synthesis of a new DNA strand. It is semi-conservative meaning each strand of existing DNA is conserved and serves as a template for new strands.
Define replication fork.
It is the Y-shaped structure where replication occurs after DNA unwinds. There are 2 of them, each moving in the opposite direction away from the origin.
Define replisome
It is a multiprotein molecular machinery that carries out the replication of DNA. In other words, it is all the enzymes that replicate DNA.
State the functions of replisome components: DnaA, DnaB, DNA polymerase III, DNA Primase, DNA gyrase, DNA ligase, and SSB Proteins.
DnaA is the first protein to bind oriC (the origin of replication) and breaks the hydrogen bonds to open it.
DnaB is a helicase that unwinds and unzips the DNA to make the replication fork.
DNA gyrase relieves the tension from the unwinding DNA by making cuts into the DNA.
Single-strand DNA-binding proteins (SSB) stop the DNA from retwisting.
DNA primase makes RNA primers, which are the complementary strands.
DNA polymerase III synthesizes DNA from 5’ to 3’ (extends the DNA)
DNA Ligase seals the DNA by making phosphodiester bonds.
Explain the molecular events that take place during the initiation phase of bacterial replication.
DnaA binds to the oriC to break the hydrogen bonds to open it and recruits DnaB and DnaC complexes. DnaB unzips/unwinds DNA to make the replication fork. DNA gyrase then relieves some of the tension from unwinding by making cuts into the DNA. SSB then binds to the 2 DNA strands and stops DNA from retwisting. After this, DNA primase creates the RNA primers, (which provides the necessary 3’ -OH) that create the complementary strands.
Explain the molecular events that take place during the elongation phase of bacterial replication.
DNA polymerase III synthesizes DNA from 5’ to 3’. The leading strand, the strand of DNA that is made continuously at the replication fork, is made in one long strand and extends towards the replication fork at 5’ to 3’. The lagging strand of DNA is made discontinuously at the replication fork and is made in short Okazaki fragments which extend away from the replication fork at 3’ to 5’. The exonuclease removes the RNA to form DNA and DNA ligase binds the 2 pieces of DNA from the lagging strand by creating phosphodiester bonds.
Explain the molecular events that take place during the termination phase of bacterial replication.
The replisome reaches a termination sequence (ter) and dissociates from DNA. In other words, the DNA links at the fork and falls off.
During initiation of DNA replication, the enzyme _______ unwinds and unzips DNA.
DNA helicase
During elongation in DNA replication, the enzyme ______ functions to add nucleotides to a growing DNA strands.
DNA polymerase III
What is true about DNA replication?
DNA is made in small Okazaki fragments on the lagging strand.
DNA replication is continuous on the leading strand.
What events are correctly paired with the stage in DNA replication in which it occurs?
Initiation: DnaA binds to the origin of replication to recruit DNA helicase (DnaB).
Elongation: Nucleotides are added to growing DNA strands.
Termination: Replication machinery dissociates from DNA.
How is DNA structured?
It is made of monomers called deoxynucleotides, that has nucleotides bound together by phosphodiester bonds, which form the 2 antiparallel, double helix. The strands are held together by hydrogen bonds between complementary bases (A=T, C=G).
What are the 3 parts of Deoxynucleotides?
Phosphate group
5-carbon sugar (deoxyribose)
Nitrogen base
How many nitrogen bases are there and how are they grouped?
There are 4 nitrogen bases with 2 groups:
Purines that have Adenine (A) and Guanine (B)
Pyrimidine that has Cytosine (C) and Thymine (T)
What are phosphodiester bonds?
A bond between the phosphate group of 1 nucleotide and the sugar of another.
What are templates called for DNA?
parent strands
How many hydrogen bonds do A=T and C=G make and what does that mean?
A=T makes 2 bonds while C=G makes 3. The 2 bonds of A=T are easier to break while the 3 bonds are tighter.
Define promoter
It is a binding location for RNA polymerase and its upstream before the coding region.
Define coding region
A coding region is a sequence of nucleotides that specifies the amino acid sequence in the protein gene product. It always starts with AUG and ends with a STOP codon.
Define terminator
It is a signal for RNA polymerase to stop transcription and it is downstream after the coding region.
Name the enzyme that synthesizes mRNA from a DNA template during transcription.
RNA polymerase
List the function of sigma factor and rho factor.
sigma factor is the initiation of transcription binding the promoter during transcription. rho factor is involved in the termination of transcription.
Explain the molecular events that take place during the initiation phase of bacterial transcription.
RNA polymerase binds to the promoter region, which is positioned by the sigma factor, DNA polymerase unwinds the DNA then the sigma factor falls off to begin elongation.
Explain the molecular events that take place during the elongation phase of bacterial transcription.
This happens in the transcription bubble. As RNA polymerase is reading the bases the complementary bases are then added, and 20 nucleotides are added to a piece of RNA. This is the creation of RNA. For the base paring, the complementary strands are A=U and C=G.
Explain the molecular events that take place during the termination phase of bacterial transcription.
RNA polymerase stops moving and the rho factor disassociates the polymerase at the end, which pops the polymerase off the DNA.
What is a gene?
a nucleotide sequence that codes for one or more functional products
Which part of the RNA polymerase enzyme has no catalytic activity but functions in binding to the promoter during transcription?
Sigma factor
The coding region of a gene is "upstream" of the terminator. Conversely, the terminator region is said to be "downstream" of the coding region. What structures is/are upstream of the coding region of a gene?
Promoter, RNA polymerase binding site, and Leader
What is the order of bacterial transcription?
Sigma factor binds to RNA polymerase. The now Active RNA polymerase binds to the promoter region of a gene. RNA polymerase unwinds a small region of DNA to create a transcription bubble. RNA polymerase begins synthesizing a new RNA strand by adding complementary nucleotides to build a piece of mRNA. Then RNA polymerase reaches the terminator region of a gene and dissociates from DNA and releases mRNA.
What are the 3 types of RNA and what are their functions?
Messenger RNA (mRNA) carries genetic information from DNA to the ribosome. Transfer RNA (tRNA) is an adaptor between amino acids and the codons in mRNA. Ribosomal RNA (rRNA) provides structural and catalytic components of ribosomes.
What is transcription?
The synthesis of RNA under the direction of DNA. DNA is synthesized into RNA.
In bacteria how are genes organized and what does that mean?
They are organized into operons, which are genes for proteins involved in the same processes that are located close together and are under the control of the same promoter. This also helps the bacteria save space in their chromosome by making them smaller.
Bacterium G has a mutation in the gene that encodes rho factor (ρ). How will this affect initiation of transcription?
It won’t. Rho factor is needed for termination of transcription, not initiation.
Define start codon, stop codon, and chaperone.
START codons are needed to begin translation (AUG/MET). STOP codons end translation (UAA, UGA, UAG). Chaperones are proteins that help proteins fold by preventing misfolding and correcting misfolding.
Explain the molecular events that take place during the initiation phases of bacterial translation.
START codon in mRNA binds to the 30 ribosomal subunit, 50s ribosomal subunit binds to the 30S subunit to form an active complex.
Explain the molecular events that take place during the elongation phases of bacterial translation
This is the creation of proteins stage. tRNA comes into the A site, then the P site, it creates a peptide bind, causing it to shift down, then it goes to the E site and grows.
Explain the molecular events that take place during the termination phases of bacterial translation.
The ribosome reaches a STOP codon like UGG and dissociates from mRNA and the new polypeptide.
What are codons?
Triplets of nucleotides, each triplet encodes 1 amino acid.
What are amino acids linked together by?
peptide bonds
The _____ is used by pathogens to "inject" proteins directly into eukaryotic host cells and cause disease.
Type III secretion system (T3SS)
Which system translocates unfolded proteins across the plasma membrane?
Sec system
_____ is a molecular chaperone that physically separates misfolded proteins from other cytoplasmic proteins and gives them a place to uncoil and refold.
GroES/GroEL
The _____ of the ribosome receives tRNAs carrying amino acids that will, ultimately, be added to a growing protein.
A site
Explain the events that take place in bacterial translation.
Small ribosomal subunit binds to mRNA and reaches START codon (AUG/Met). A large ribosomal subunit (50S) binds the small ribosomal subunit (30S) to form an active complex. tRNAs bring the appropriate amino acid to the A site of the ribosome. A peptide bond forms between amino acids at the A and P sites of the ribosome. Empty tRNA exits the ribosome from the E site. Ribosome reaches a STOP (UAG, UAA, UGA) codon. Release factor binds to A site. Proteins, mRNA, and ribosomes are all released.
Tetracycline (TE) is an antibiotic that works by binding to the A site of the ribosome. How would this slow/stop translation?
Incoming tRNAs carrying amino acids must bind to the A site.
What is the difference between a one-step and a two-step secretion system?
In one-step, proteins are secreted from cytoplasm to the environment in one step. In two-step, proteins are moved in two steps: first from the cytoplasm to the periplasmic space, and second from the periplasmic space to the environment.
Why do Gram-negative bacteria need specialized secretion systems where Gram-positive do not?
Gram-negative bacteria have 2 membranes that proteins must get through to be secreted, and proteins need special systems to pass through the outer membrane. Gram-positives do not have an outer membrane.
what is tRNA and what does it contain?
an adaptor between amino acids and mRNA codons. It contains anticodons, sequences commentary to the codon in mRNA, and corresponding amino acids.
rRNA that makes up ribosomes have what types of functions?
ribosome structure
16s rRNA needed for initiation of translation
23s rRNA catalyzes peptide bond formation
What are the 3 sites on the ribosome that bind to tRNA?
A site binds incoming tRNA carrying the next amino acid to be added to the growing peptide chain.
P site binds tRNA to which the growing polypeptide is attached.
E site binds departing tRNA
Know how bacteria can increase the efficiency of gene expression as compared to eukaryotes.
Bacteria can transcribe and translate in the same cellular compartment, which makes the process of gene expression to occur quickly.
Describe the structure and basic function of the following Secretion Systems: Type II (T2SS), Type III (T3SS), and Type VI (T6SS)
Type II (T2SS) is an example of 2-Step secretion (it is 2 proteins) that only happens in Proteobacteria and secretes degradative enzymes which degrade our tissues. Type III and VI are examples of One-Step secretion systems (1 big protein); Type III secretes proteins that promote pathogenesis by puncturing itself in a cell. Type VI waits ready to go and tries to wait for a cell that’s close to secrete its toxins into. This attacks both bacterial and eukaryotic cells.
Describe protein folding and its function.
Proteins adopt adaptational structures, which are coded in the amino acid sequence. The misfolded protein can be non-functional or toxic to the cell. Chaperons help these proteins fold by preventing misfolding and correcting misfolding.
What are the 2 types of chaperons and describe them?
The first one is Trigger factor. It is associated with growing polypeptides on the ribosome. It holds onto the amino acid chain so it’s not free-floating around in the cytoplasm, which helps stop misfolding. The second is GroES/GroEL, which is a cage-like structure that gives proteins a place to fold.
What is translocation?
the movement of protein from the cytoplasm to or across the plasma membrane.
What is secretion?
the movement of proteins from the cytoplasm to the external environment
What are the 2 systems of translocation and describe them?
Sec system which moves unfolded protein across the membrane. Single peptides in polypeptide bind to signal recognition particles, the SRP brings the whole ribosome to the membrane and the Sec proteins form a channel where the growing polypeptide can move through.
Tat System moves folded proteins across the membrane, by chaperons binding a signal peptide and bringing the protein to the Tat complex, the PMF (proton motor force) then drives the movement of proteins through the Tat channel.
Genes that are expressed continuously are said to be _____.
constitutive
Transcription will be turned OFF if _____.
An activator protein in the presence of an inhibitor cannot bind to the activator binding site of a repressible gene.
The _____ gene in the lac operon encodes the enzyme β-galactosidase, which breaks down the sugar lactose.
lacZ
In regards to the lac operon, which of the following occurs when lactose is not present?
lac repressor protein is active.
What characteristics is/are shared by the lac and trp operons?
Both operons utilize repressor proteins. Both operons negatively control gene expression.
In the lac operon, does allolactose act as an inducer or a corepressor? How do you know?
It is an inducer; allolactose changes the shape of lac repressor protein —> repressor protein becomes unable to lac operator in DNA —> turns off repression —> turn transcription ON
If it were a corepressor, allolactose would promote binding of the lac repressor protein to the DNA —> turn transcription OFF.
In this scenario will transcription be turned ON? Explain why or why not. An activator protein in the presence of an inducer binds to the activator-binding site of an inducible gene
ON; inducer changes shape of activator protein so it can bind DNA —> activator protein binds to activator-binding site —> turns transcription of an inducible gene ON
In this scenario will transcription be turned ON? Explain why or why not. A repressor protein in the presence of an inducer is unable to bind the operator of an inducible gene.
ON; inducer changes the shape of the repressor protein so it can no longer bind to DNA —> repressor protein no longer represses inducible gene —> turns transcription ON
Define constitutive gene, inducible gene, and repressible gene.
Constitutive genes are genes that are continuously expressed, they are always on and are “Housekeeping genes” Examples of this are ATP-making and respiration genes.
Inducible genes are genes that turn ON in response to something.
Repressible genes are genes that turn OFF in response to something.
Explain how activators and repressors proteins exert control over gene expression
Activator proteins ACTIVATE transcription by binding to the DNA which is a positive control. They are activated by inducers and inactivated by inhibitors.
Repressor proteins bind DNA to turn transcription OFF, which is a negative control. It is activated by corepressors and inactivates by inducers.
Discuss the role/s of inducers, inhibitors and corepressors in different types of
regulation.
Allosteric regulation regulates by changing the shape of regulatory proteins. The inducers for Active proteins can change the shape of activator proteins, which helps them bind to DNA. Inhibitors for Active proteins bind to the protein which turns transcription off. For the repressor proteins, the corepressor binds to an active repressor to turn transcription off. Inducers for Repressor proteins, inactivate repressor, which then turns on transcription.
What are the genes encoded by the lac operons?
LacZ which breaks down lactose. Lac-Y which brings lactose into the cell and LacA, which helps with the breakdown of a lactose.
Explain what happens to regulate the lac operon when lactose is present/absent
If there’s no lactose to take into the cell then these genes (LacZ, LacY, and LacA) are off. Lac repressor proteins bind to DNA to cut off transcription, and the repressor proteins regulate this.
If there is lactose, transcription is ON. An inducer binds to the lac repressor which turns on transcription, which stops the blocking of the enzyme. The inducer changes the shape of the lac repressor which makes it unbind from the lactose.
Compare and contrast the lac operon and the trp operon
Both operons utilize repressor proteins. Both operons negatively control gene expression. Lac operons utilize lactose as an energy source while Trp operon’s function is to make the amino acid tryptophan. For low tryptophan an inducer regulates it, and DNA polymerase turns it on. High tryptophan, a corepressor turns transcription off, and tryptophan turns the creation of trp off (it essentially uses itself to stop the making of tryptophan).
The type of horizontal gene transfer that allows bacteria to transfer DNA via cell-to-cell contact is _____.
Conjugation
What are the steps of F factor-mediated conjugation?
Sex pilus binds to F- cells and pulls cells close together, the secretion system is assembled, coupling factor brings DNA to the secretion system. DNA is transferred to F- cell, making it F+
The type of transduction that results from incorrect removal of a prophage from a bacterial chromosome is known as _____.
Specialized transduction
Transformation would be prevented if _____.
The cell surface receptors on cells were mutated and no longer able to bind DNA. The DNA uptake system in the membrane was mutated and non-functional. The extracellular endonuclease was inhibited, and DNA could not be cut into smaller pieces.
Define conjugation, sex pilus, transformation, transduction, and prophage.
Conjugation is the transfer of DNA by direct cell-to-cell contact (it is similar to sexual reproduction).
Sex pili are longer/wider in diameter and allow DNA to move, it is a structure needed to connect 2 cells during conjugation.
Transformation is the uptake of free DNA from the environment (usually plasmid or linear DNA).
Transduction is the transfer of genes between bacterial cells by viruses.
Prophage is viral DNA.
Describe the factors that can make bacteria naturally transformation-competent
In transformation, a bacteria takes up free DNA from the environment, usually plasmid or linear DNA. Anything picked up from transformation can be passed on to the next generation. Competency requires the uptake system and receptor. DNA that’s picked up could come from lysed/ dead cells, The receptor makes the DNA plasmid competent, DNA fragment binds to a cell surface receptor. The uptake system then brings it into the cell.
What is competency?
It is the stage of growth in bacteria (stationary phase of growth); wants to pick up more nutrients.
Outline the process of transformation.
DNA fragment binds to a cell surface receptor, an extracellular endonuclease cuts DNA into smaller fragments, one strand is degraded, and a single strand is transported into the cell, using the uptake system. The DNA strand aligns itself with a homologous region on the bacterial chromosome. The DNA strand is then incorporated into the bacterial chromosome, then the heteroduplex DNA is repaired changing lac- ro create a lac+ gene.
How can cell be made artificially competent in the lab?
You can use a calcium chloride treatment and then heat shock or electrical shock to infuse sample DNA into bacteria. This procedure then can be used to produce proteins for commercial use like making insulin, growth hormones, or steroids.
Compare the function and mechanisms of generalized transduction and specialized transduction.
Generalized transduction only occurs during the lytic cycle. It transfers any part (random bacteria) of the bacterial genome, and bacterial DNA is mistakenly packaged into viral capsids during assembly. In this process, there are fewer transducing phages than specialized transduction, and there is only 1 messed up DNA. The messed-up DNA is an error on the virus’s part, which doesn’t affect the bacteria since it produces many normal phages ¾ are normal, which is advantageous since it allows the bacteria to mix together.
Specialized transduction occurs during the lysogenic cycle. It transfers specific parts of the bacterial genome by improperly cutting out viral DNA, which creates phages that become both viruses and bacterial DNA, 4/4 are completely useless as phages can only be used for transduction. This dramatic error prevents itself from being replicated again.
Why is HGT so important to bacteria?
it increases genetic variation in bacteria, since bacteria can’t partake in sexual reproduction, then they mix genes through horizontal gene transfer, two independent mature organisms that transfer genes or the transfer of genes between bacteria.
Some bacteria exhibit two-phase growth when exposed to two nutrients and utilize one nutrient first before altering their metabolism to utilize the second. This is known as _____.
Diauxic growth
In a two-component system, the _________ auto-phosphorylates to transmit a signal from the extracellular environment to the intracellular environment.
Sensor kinase
The OmpC and OmpF proteins who expression is controlled by the EnvZ/OmpR system are _________.
Porin proteins
Explain EnvZ/OmpR system.
The EnvZ/OmpR system is an example of a two-component system. EnvZ is the sensor kinase. OmpR is the response regulator.
Explain chemotaxis in E. coli.
CheZ inactivates CheA by dephosphorylating it. CheY is a response regulator that causes a change in the rotational direction of bacterial flagella. CheA is a sensor kinase that auto-phosphorylates and phosphorylates CheY.
Define phosphorelay system.
a set of proteins involved in the transfer of a phosphate from one protein to another.
Define diauxic growth.
biphasic growth response, the use of 2 sugars and different phases
Describe the functions of both ‘components’ in a two-component system
Sensor kinase is a protein that can communicate information from outside to inside bacteria; it works by giving a phosphate group to the response regulator, which mainly transcribes factors such as proteins that can increase or decrease transcription. The sensor kinase EnvZ can sense osmolarity (salt in the environment). The response regulator OmpR is an outer membrane protein response that is important for transcription.
Explain the mechanism and ‘big picture’ outcome of the EnvZ/OmpR system
The outcome is it turning genes on and off.
In low salt environments the EnvZ is turned OFF, the OmpF is higher since it allows more things to pass through with it having a bigger pore, since it is a low salt environment it takes a lot of the salt from the environment in, which is why it needs the OmpF. The OmpC is off since the pore is smaller and useless in this type of environment, and the response regulator OmpR is turned into OmpF.
In high salt environments EnvZ is activated and ON, its job as a kinase is to phosphorylate stuff like OmpR; OmpR then stops working to make OmpF to OmpC. The OmpC takes in a little bit of water and salt so its OmpC is high and OmpF is low.
Describe the mechanism and advantages of diauxic growth in E. coli.
If E. coli grows in the presence of glucose and lactose, it will start by using glucose (which requires the Kreb Cycle, a complex process) it will then switch to lactose (which requires a lac operon), at the stationary phase when glucose is gone.
Outline the mechanism of how E. coli senses and, ultimately, moves toward a chemoattractant.
In the presence of a chemoattract bacteria runs towards it. If CheA isn’t working in the presence of a chemoattractant then the flagella will “run” CCW. If CheA is active then it phosphorylates CheY, a response regulator, which causes the flagella to rotate CW “tumble”. CheY is then inactivated (de-phosphorylated) by CheZ to stop tumbling to move CCW “run”. This is all directed movement going towards a chemoattractant.
What type of complex processes can bacteria perform?
Endospore formation, chemotaxis, and metabolism
What is global regulatory system?
a 2-component system, 2 proteins sensor kinase and response regulator, that affects many genes at the same time
What are MCPs?
a chemoreceptor that binds chemoattractant
Episome
a plasmid that can exist within or outside of a bacterial chromosome
Note
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