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Exam III Review

Page history last edited by Lwentz 13 years, 2 months ago

This is an interactive review sheet that encourages you to interact with the material and draw out the critical information.  There are several ways to contribute:

 

  • Add an important vocabulary word and its definition to the list from a specific lecture.  These are anything you feel is essential to the understanding of the material including the names and functions of proteins, types of chemical bonds, names of processes, etc.

  • Add an answer to one of the concept questions

  • Edit and expand on another student's posting

  • Add a link to another website, a video on You Tube, or any other teaching material on the web that you think enhances your understanding of the material.

 

As I stated in class, 8 points of your exam grade is dependent on your participation in this dialog about the material.  I will use the following rubric for grading: 3 points for adding and defining one vocabulary word, 3 points for participating in the answering of one of the concept questions, 2 points for editing and expanding on another student's posting (edits must be more than just removing a word or adding a word, it must have an impact on the material discussed)

 

Contributions to this review must be completed by 5 p.m. on March 31st (the Tuesday before the exam)

 


Lecture 10 Prokaryotic Transcriptional Machinery

 

Important Vocabulary

 

Core polymerase: the part of Polymerase that actually makes the RNA.  Consists of 5 subunits and has catalytic activity, but minimum transcription activity.  It cannot recognize DNA by itself (it needs a sigma factor).

 

Holoenzyme- consists of the core polymerase and the sigma factor.  This combination brings the core polymerase to the DNA.  The enzyme needs a 3' OH end for addition.  New nucleotides are attached at the 3' OH end.  The enzyme needs no primer, as it utilizes a Ribo-Nucleotide TriPhosphate with a 2' OH end.  Abortive initiation occurs where the complex "revs its engines" and gets ready to go.  When the sigma factor undergoes a change in formation, the polymerase is released and begins copying leaving the rest of the proteins behind.

 

Polycistronic- An mRNA that encodes for two or more proteins or a set of genes.

 

Monocistronic- An mRNA that encodes for one promotor which controls only one gene.  In eukaryotic cells.

 

Promotor Strength- number of transcripts initiated at a given time.  The two kinds of promoter strengths are strong and weak.  A weak promoter  causes little RNA to be made due to a weak interaction with the holoenzyme which causes it to be unstable.  However, a strong promoter causes lots of RNA to be made due to a strong interaction with the holoenzyme which causes it to be stable.  Consensus is also important.  If the promotor has a good match to the consensus sequence, then a strong interaction results.  If the promotor does not match the consensus sequence, then a weak interaction results.  The number of bp's inbetween -35 and -10 also affects the strength. If there are too many base pairs (over 19) or too few (under 17), the -35 and -10 regions will not be aligned on the same side of the DNA, which causes the sigma factors to be unable to interact with the regions.

 

Sigma Factor - is used in prokaryotic transcription and helps initiate the process of transcription.  The sigma factor is an initiation factor that binds RNA Polymerase to promoter sequences on a gene.  The promoter regions that it recognizes are -10 and -35.  These promoter sequences are 6 base pairs long.  The core Polymerase and the sigma factor join to form a holoenzyme which binds to the DNA and melts the DNA open to make a small transcription bubble within the holoenzyme.  Without the sigma factor, RNA Polymerase cannot bind to a strand of DNA and thus transcription cannot take place. 

 

Transcript- The messenger RNA chain produced by transcription that has a nucleotide sequence exactly complementary to the strand of DNA used as the template.  The nucleotides used to create the transcript are the same as DNA except for the exception of Uracil replacing Thymine.

 

Consensus Sequence- the ideal sequences at the -35 and -10 regions on the DNA molecule; the closer the DNA sequence is to the consensus, the stronger the promotor.  This sequence is where the RNA polymerase binds to the DNA and melts open the helix which allows transcription to take place beginning at the +1 site.

 

UP element  -  A promoter element characteristic of certain strong promoter regions; where RNA polymerase likes to bind. Consists of an AT rich region. Increases promoter strength, but not always present.

 

Terminator sequence: an inverted repeat sequence causing the formation of a hairpin in the RNA strand, which causes the RNA to fall off (Rho-Independent Termination)

 

 

An overview of prokaryotic transcription:http://www.ncbi.nlm.nih.gov/books/bv.fcgi?rid=cooper.section.964 

 

Prokaryotic RNA Polymerase: http://homepages.strath.ac.uk/~dfs97113/BB310/Lect13/lect13.html

 

 

Important Concepts

 

What are the main components of a transcriptional unit?

The Holoenzyme  consists of the Core + the Sigma Factor. The Core Polymerase is 5 subunits which have catalytic activity. Alone, the Core has minimal transcription activity because it is unable to ID DNA alone. The Sigma Factor brings the Core to the DNA. The Holoenzyme then moves 5' --> 3' with no primer.

 

What are the three parts of the transcriptional cycle?

The three parts are 1.) initiation 2.) elongation 3.) termination.  Initiation consists of the RNA polymerase binding the promoter and then melting open the DNA forming the initial transcribing complex.  Elongation consists of the DNA being transcribed into the RNA strand.  Termination occurs when the polymerase terminates and releases the RNA strand.  Termination can be either Rho-independent or Rho-dependent.

 

How is RNA Polymerase different from DNA Polymerase?  How are they similar?

Although RNA and DNA polymerase catalyze essentially the same reaction, there are some important differences between the two enzymes. RNA catalyzes the linkage of ribonucleotides, not deoxyribonucleotides.Unlike the DNA polymerases involved in DNA replication, RNA polymerases can start an RNA chain without a primer. This difference may exist because transcription need not be as accurate as DNA replication. Unlike DNA, RNA doesnt store genetic information permanently in the cell. RNA polymerases make one mistake for every 100,000 nucleotides copied into RNA compared with and error rate for direct copying by DNA polymerase of about one mistake per 10^7 nucleotides. Altough the RNA polymerases are not as accurate as the DNA polymerases, they also have a proofreading system.If the incorrect nucleotide is added to the growing RNA chain, the polymerase can back up, and the active site of the enzyme can form an excision reaction that mimics the reverse of the polymerization reaction.  Both RNA polymerase and DNA polymerase add in the 5' to 3' direction.

 

 

How is the strength of a promoter determined?

The strength of the promoter is determined by the protein's affinity for the DNA sequence of the promoter.  The affinity of the protein to the DNA is based upon how closely the DNA matches the consensus sequence, or the ideal base pair pattern for binding.  If the promoter strength is high, a lot of RNA formation will be occuring.  If the interaction between the DNA and the holoenzyme is weak, then there will be less transcription. The number of nucleotides between the sequences is important as well.  If you add or removes bases between the sequences, the sequences will rotate to different positions on the helix and the sigma factor will have trouble interacting with both at the same time.

 

What is the function of the sigma factor?

The sigma factor is a DNA binding protein which enables RNA polymerase to bind to promoters on DNA.  It is also required for the melting open of DNA. In order to initiate transcription in prokaryotes, core RNA polymerase and the sigma factor form a holoenzyme. The sigma recognizes the promoter sequence by making specific contacts with portions of the base. The sigma factor is a detachable subunit and is largely responsible for the ability to read signals in the DNA  that tell it where to begin transcribing.  After the polymerase is recruited and stabilized the role of the sigma factors is completed.  As the polymerase moves down the DNA, it leaves the sigma factors, along with the rest of the holoenzyme, behind. 

 

How is transcription terminated in prokaryotes?

Termination can either be Rho independent or Rho dependent.  In Rho independent termination, RNA Polymerase synthesizes nucleotides until the enzyme encounters the terminator signal in the DNA.  The termination signal consists of a string of A-T nucleotide pairs preceded by a two-fold symmetric DNA sequence that folds into a hairpin structure through Watson-Crick base pairing.  At the end of this hairpin structure a poly "U" tail is formed by the repeated "A" sequence on the template strand.  The strong G-C bonds in the hairpin structure cause the polymerase to pause while the weak poly U sequence is transcribed. This pause and the weak U-A binding causes the release of the RNA transcript and dissociation of RNA. The polymerase is therefore released from the DNA.

 

Rho dependent termination involves a Rho factor to cease transcription.  The RNA polymerase transcribes a rho-utilization (RUT) site which is then recognized and bound by the Rho protein. The Rho factor acts as a helicase using ATP hydrolysis to "pry" the RNA off of the polymerase.  It runs along the newly synthesized RNA strand toward the 3' end where the RNA and DNA template strand form a transcription fork. It is believed that the Rho protein is able to "catch up" to the polymerase when the polymerase pauses at the Rho-termination site, and when the Rho protein reaches the polymerase it uses its helicase activity to cause the polymerase to dissociate. 

 

 


Lecture 11 Phage: The Genetic Switch

 

Important Vocabulary

 

Lytic Cycle- When discussing λ phage infected bacteria, this is one of two processes that can occur. In the lytic cycle, the repressor gene is off, the cro gene is on and phage genes are activated.  The λ chromosome is replicated numerous times, new head and tail proteins are synthesised, and new phage particles are formed within the bacterium until the cell lyses, releasing progeny phage.

 

 λ repressor- regulates whether a gene is turned on or off.  It is a negative regulator that "turns genes off" ( it turns the cro protien off and prevents the binding of polymerase) and it is also a positive regulator that "turns genes on" ( it turns itself on to "touch" the polymerase stabilizing it). This protien has two domains, one on either ball of it's dumbbell shape. Domains: N-terminal Domain is the end that binds to the DNA. C-terminal domain is the Dimerazation domain, which binds another copy of itself to form a dimer. Dimers are found bound to DNA in cells.

 

Cro Protein-  is the DNA binding/dimerization domain.  It has a high affinity for Or3 and also shuts off the lambda repressor protein. It is a negative regulator and prevents the binding of polymerase.

 

Lysogenic Cycle: This is when the Viruses DNA is incorportated in the bacterias DNA.  The λ repressor is on and the cro gene is off.  Once the cell starts dying the λ repressor will be turned off and the cro gene on and the cell will go into the Lytic stage.

 

cI: Lambda repressor.  Can bind OR1, OR2, and OR3.  Affinity: OR1=OR2>OR3.  Binding at OR1 increases the affinity to OR2.  When bound to OR1 and OR2, promotes its own transcription by increasing RNA polymerase affinity and blocks cro transcription.  When bound to OR3 at high concentrations, inhibits own transcription by blocking RNA polymerase binding region.  cI may be destroyed by activated recA.

 

CII:  The protein largely responsible for determing if the λ phage goes into the lysogenic or lytic pathway.  CII activity is controled by environmental factors, which affect the bacteria cell.  If the bacteria is healthy, in a rich medium, then it will produces high levels of protease.  These proteases are going to destroy the CII protein made by λ phage.  When CII is destroyed, the system goes into the lytic cycle.  On the other hand, if the bacteria is in a poor medium and is really hungry, then it won't be able to make proteases.  CII is NOT degraded (with the help of CIII, which protects CII).  With CII present, lysogeny is established.  

 

Operator: the specific DNA sequence bound by regulators.  Repressors bind to this specific site and inhibit transcription by blocking the DNA strand so polymerase cannot bind.

 

OR2: (operator)prevents polybinding to Pr; negative regulator of Cro; stablizes polymerase on Prm; positive regulates itself 10x.

 

PRM - The regulatory region of bacteriophage lambda contains two promoters, PRM and PR.  PRM contains part of the OR2 and the OR3 operators.  This promoter points polymerase leftward and has a weaker promoter strength than PR.

 

PR - The regulatory region of bacteriophage lambda contains two promoters, PRM and PR.  PR contains part of the OR2 and the OR1 operators.   This promoter points polymerase rightward and has a higher promoter strength than PRM.

 

Important Concepts

 

What is negative control of a promoter?

Negative control means the protein binds to DNA to inactivate expression of itself or another gene. 

 

Example: Negative control occurs when the repressor is located at OR2 (part of the right operator) in turn turning off the cro gene by preventing binding of RNA polymerase to PR (a promoter that points polymerase rightward). The repressor covers part of the DNA that polymerase must see to bind to PR. Negative control is partly responsible for maintaining the lysogenic state.

 

What is positive control of a promoter?

Positive control can be seen when looking at a repressor located at OR2. This repressor helps RNA polymerase bind and transcribe at PRM (the cl promoter which points polymerase leftward), this is responsible for helping transcription of cl in a lysogen. This repressor can help increase transcription of its own gene. This repressor represses PR by excluding binding of RNA polymerase to that promoter but it encourages polymerase to begin transcription at PRM (it prevents transcription to the right but aids transcription to the left).

 

What is an operator?

     An operator is a regulatory unit of an operon to which activators and repressors bind to effect the transcription of genes in the operon (DNA sequence that is bound by regulators).  In the lambda phage, the operators are OR1, OR2, and OR3.  These are the three sites to which cI and cro proteins are able to bind, and depending on the protein and operator(s) to which that protein is bound, either cro or cI genes are expressed.

 

What is the lambda repressor and how does it function to shut down phage gene expression?

The lambda repressor can act as both a negative and positive regulator.  It acts as a negative regulator by turning off or inactivating certain targeted genes.  It acts as a positive regulator because it also helps to turn itself on or activate itself by stabilizing the RNA polymerase to the Prm promoter.  It is able to shut down phage gene expression by binding to Or1 and Or2 located on the DNA of the gene.  When repressors are bound to the Or1 and/or Or2 sites, they force the RNA polymerase to bind to the Prm and transcribe to the left.  Repressor genes are located to the left of the operon, so in this way it can be said to positively regulate transcription of itself.  In this way it also shuts down phage gene expression because Prm is a weak promoter and transcription occurs at a very low rate when RNA polymerase is bound to it.  Thus when transcription occurs at a very low rate, genes that would be transcribed and translated to make proteins are not expressed (are "turned off").

 

How does repressor activate its own transcription?

When repressor binds at O2, it interacts with RNA polymerase and helps the enzyme bind and begin transcription at Prm.  The repressor gene is located to the left of the operators and the Prm promoter (a weak promoter) initiates transcription to the left.

An operon contains three repressor binding sites (operators); Or1, Or2, and Or3 which lie between two promoters. We also know that an amino acid-terminal domain promotes transcription in the left direction once it is bound to the Or2 site.  This occurs by directly coming into contact with the RNA polymerase to stabilize it on the Prm promoter.

 

How does repressor prevent cro expression?

The repressor, cI, has high affinity for OR1.  When it binds to OR1, it prevents RNA polymerase from binding to the Pr promoter.  The Pr promoter is a strong promoter that initiates transcription to the right toward the cro gene site.  Thus, cro can not be expressed in the presence of cI repressor.

 

How does cro turn off repressor expression?

It turns off repressor expression by having a high affinity for and binding to Or3 which in turn shuts off repressor expression by blocking the binding site on the Prm promoter.  The Cro also has no cooperative binding.

 

What is cooperative binding?

In cooperative binding, the binding of one molecule facilitates the binding of another molecule, increasing stability due to the interactions between the two molecules. It increases the repressor-opertor affinity.  In λ repressor there is low affinity when the protein binds to OR2 but when another protein binds to OR1 there is high affinity which further stablizes the polymerase proteins because they are binding at the C terminal end and the N termanal in.

 

How does cooperative binding explain how Operator 2 fills quickly after Operator 1 is bound?

Or2 fills in quickly after Or1 because of cooperative binding. The protein has a naturally high affinity for Or1 and makes a tight interaction with it. The protein does not have as high of an affinity for Or2 as Or1 but needs to keep being filled at a level to keep the genes being repressed. Cooperative binding increases the affinity for the protein to the site because of the close protein interaction between the two dimmers. Or3 does not have this protein interaction, therefore it will remain relying on a week affinity for binding.   

 

When bacteria are irradiated with UV light, how is the switch flipped so that the phage becomes lytic?

When the bacterai are irradiated with UV light, almost every lysogen in the population will lyse and produce a new crop of Lambda.  The UV light turns on previously inert phage genes and lytic growth ensues.  Agents such as UV lgiht induce lytic growth in lysogens by damaging the host DNA.  Then, the phage chromosome uses bacterial enzymes to sense the impending death of the cell and then enters the lytic pathway.  It senses this condition because when the bacteria is under stress the Rec A protein becomes a protease.  This protease attacks the lamda repressor dimers, destroying them (they turn into monomers).  The amount of lamda repressor bound to OR1 and OR2 therefore decreases and the transcription of repressor also decreases.  This triggers the expression of cro.

 

What is the biochemical basis for repressor preferring operator 1 while cro prefers operator 3?

Or1 and Or3 are essentially the same sequences except for minor nucleotide sequences. These nucleotide differences are the reason why repressor preferrs to bind to Or1 while cro prefers to bind to Or3. Both the repressor and cro are made up of helixes. The role of the helixes are to make contact with DNA, each helix has an affinity for a a specific sequence. The repressor is able to make more contacts with OR1 making it more stable. Cro is able make more contacts with OR3 making that a more stable operator.  Repressor and Cro are composed of different amino acids.  Certain AA have a higer affinity for binding to certain bases than others.  Repressor and Cro share similar AA (gln, ser) which bind to the same sequence in either OR1 or OR3.  This explains why repressor and cro can bind to both operators, however, repressor forms the most/strongest protein-DNA interactions at OR1 and Cro forms the most interactions at OR3

 

Why does the repressor promoter require positive control for high expression?

The repressor promoter requires positive control (the binding of itself to Or2 to stablize the bond between the Polymerase and the DNA) because the promotor sequence Or3 is a weak promoter. There is a two base difference in the promotor sequence of the repressor protein "Or3" and the Consensus sequence ( the sequence of bases that has the highest bonding strength).

 


Lecture 12 Phage: Setting the Switch

 

Important Vocabulary

 

Q-Protein:

A protein that allostericly changes the conformation of the RNA polymerase so that the RNA for the Head and Tail pieces of the Phage are transcribed, when in the Lytic cycle. N-->Q-->Lysis genes transcription. Another "anti-terminator."

 

N-Protein: The Anti-terminator protein. It allows the polymerase to ignore the termination signal. Also allows all other genes to be made into long strand of RNA during very early transcription of a phage.

 

Genetic Cascade: Event where a set of genes are turned on and from that set of genes there are one or more specific genes that turn turn on a second set of genes which also has one or more genes that turn on a third set of genes and so forth. This process is used by all forms of life, and is an important factor in the process of determining whether a cell will undergo the lytic or lysogenic pathways.

 

CII: Also known as "the decider", this protein is important in the genetic cascade process (a positive regulator), and levels of the CII protein dermine whether a cell will undergo the lytic or lysogenic pathways. If the cell is healthy and in a nutrient rich environment, CII levels will be low, and will follow the lytic pathway. If the cell is starved or in a low nutrient environment, CII levels will be high and the cell will follow the lysogenic pathway.

 

cos site: complementary bases at both ends of the linear bacteriophage DNA ("sticky ends") that allow the DNA to become circular when it enters a bacterial host. The circular structure enables the genome to be continuously replicated so that many complete genome sequences are strung together. The cos sites show the phage where to cut the genomes so that they can be individually packaged into the new bacteriophages.

OR1: Negative regulator of Pr(a strong promoter) aka the cro protein preventing transcription when bound. Lamda repressor binds in major groove of this origin.

Important Concepts

 

How do reporter plasmids work?

These plasmids are named "reporters" because they report on promoter activity. Reporter plasmids are used to monitor the amount of B Galactosidase produced by the promoter on the lacz gene. The reporter binds to the promoter and allows us to record and monitor the activity of lacz by turning on and off gene expression of the repressor.

 

What is a signaling cascade?

A signaling cascade is a series of reactions that occur when one gene is turned on which subsquently turns on other genes. These genes may then turn on other genes and so on.

 

What are the earliest expressed genes upon infection by lambda phage?

Cro and the N protein are the earliest to be turned on. Then a burst of expression primes virus to choose between lysis or lysogeny.

 

What does the N protein do?

The N protein is an antiterminator, and it allows polymerase to ignore termination signals. When the N protein is expressed, a long RNA molecule is made with all genes. During the lytic cycle, once N is activated, it facilitates the activation of Q (leading to lysis). During the lysogenic cycle, once N is activated, it facilitates in the activation of cII.

 

What genes are on when the phage decides to grow lytically?

Cro and Q are on during lytic transcription. Q allows transcription at P'r to take place, which codes for the late genes - head,tail, and lysis.

 

What genes are on when the phage decides to grow lysogenically?

cI (repressor), cII, and cIII are on during lysogenic transcription. 

 

How does the cII protein help decide which path the phage takes?

The amount of cII protein produced is "the decider". Healthy bacteria have rich media with high levels of proteases which results in low levels of cII protein because proteases "chew" cII proteins. This inactivity of the cII protein causes the phage to undergo lysis. Starved bacteria; however, are low in nutrients and have decreased levels of proteases and therefore levels of cII protein remain elevated. This activity of the cII protein causes the phage to undergo lysogeny. This makes sense because starving cells lack the nutrients needed to build new phages.

 

What is the benefit for linear bacteriophage to become circular? 

Being circular in structure allows for the continuous replication/transcription of the genome so that many genome sequences are strung together.

 


Lecture 13 The Lactose Operon and Transcriptional Control

 

Important Vocabulary

 

allosteric regulator- molecule that affects the affinity of a protein for some binding site by interacting with the protein and changing its shape.  ie. allolactose is an allosteric regulator because it binds with the lac repressor changing its shape and decreasing its affinity for the lac operator

 

cAMP- this molecule allosterically regulates CAP binding to the Lac Operon Promoter; cAMP binds to CAP forming a complex which is then activated and able to bind to DNA.

 

constituitive level- low level of expression. Also known as the basal level of expression characterized by the low level of expression of RNA polymerase.

 

CAP (Catabolite Activator Protein)- DNA binding protein which acts as a catabolic activator and is allosterically regulated by CAMP.

                                                 - activity is related to glucose levels

     At high gluclose levels, levels of CAMP are low and there is no CAP binding

     At low glucose levels, levels of CAMP are high as long as lactose is present, CAP binds to cAMP activating the operon to bind DNA, high expression

 

Mer R- acts as repressor and is allosterically regulated by the presence of mercury.  It twists DNA so that -35 and -10 are on the same side causing RNA polymerase to have trouble binding.

 

lac Operon Genes:

               Lac I = Gene for repressor protein

               Lac Z = Gene for ß-galactosidase...converts lactose into Galactose and Glucose (prefered carbon source).

               Lac Y = Gene for ß-galactoside permease...brings Lactose into cell.

 

Lac Repressor- is a DNA-binding protein which inhibits the expression of genes coding for proteins involved in the metabolism of lactose in bacteria. It operates by binding to the major groove of the operator region of the lac operon. This blocks RNA polymerase from binding, and so prevents transcription of the mRNA coding for the Lac proteins.

Glucose:  Major preferred carbon source, turns operon off with or without lactose increasing affinity of repressor to the DNA.  Important in regulating gene expression, when gone operon turns on as lactose decreases repressor affinity.

Important Concepts

 

How is the lactose operon combinatorially controlled?

 

The lactose operon consists of three structural genes,and a promoter, terminator, regulator, and operator.  The operon is regulated by several factors such as the presence of lactose and glucose.  It is combinatorially controlled in the presence and absence of the factors.  When glucose and lactose are both present basal level of transcription occurs.  When glucose is present and lactose is absent no transcription takes place.  When glucose is absent and lactose is present there is an activated level of transcription.   

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