Genetics Chapter 16
What is the regulator protein in positive control?
- In positive control the regulator protein is an activator
Riboswitches protein present
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How do operons work?
1. An operon is group of structural genes plus sequences that control transcription 2. A separate regulator gene-- with its own promoter-- encodes a regulator protein that may bind to the operator site to regulate the transcription of mRNA 3. The products of mRNA catalyze reactions in a biochemical pathway.
Levels of gene regulation
1. Compact DNA: Alteration of Structure (chromatin-- accessibility) 2. Relaxed DNA: Transcription (on, off, constitutive, where when) 3. Pre mRNA: mRNA processing (5' Cap, poly-A tail, splicing, etc) 4. Processed mRNA: RNA stability (how much mRNA/how fast degraded) Translation (how fast protein is synthesized) 5. Protein (inactive) Post translation modification (affect function of the protein ( active or not active)) 6. Modified protein (active)
Regulation of the Lac Operon
1. In absence of lactose, the regulator protein (a repressor) binds to the operator and inhibits transcription 2. When lactose is present some of it is converted into allocates which then binds tot he regulator protein making the protein inactive. 3. The regulator protein cannot bind to the operator and the structural genes are transcribed and translated (B-galactosidase, premise, transacetylase)
Catabolite repression process when glucose is high
1. Levels of cAMP are low and cAMP is less likely to bind to CAP 2. RNA polymerase cannot bind to DNA as efficiently so transcription is at a low rate.
Lactose Metabolism Process
1. Permease actively transports lactose (b/c too big to get in on own) into the cell where the enzyme B-galactosidase breaks it into galactose and glucose. 2. B-Galactosidase also converts lactose into the related compound allolactose and converts allolactose into galactose and glucose.
How do the different secondary structures form when tryptophan is high?
1. RNA polymerase begins transcribing DNA producing region 1 2. A ribosome attaches to the 5' end of the 5' uTR and translates region 1 while region 2 is being transcribed 3. RNA polymerase transcribes region 3. The ribosome does not stall at the Trp codons, because tryptophan is abundant. 4. The ribosome covers part of region 2, preventing it from pairing with 3. 4 is transcribed and paris with 3 producing the attenuator that prevents transcription.
How do the different secondary structures form when tryptophan is low?
1. RNA polymerase begins transcribing the DNA, producing region 1 of the 5' UTR 2. A ribosome attaches to the 5' end of the 5' uTR and translates region 1 while region 2 is being transcribed 3. The ribosome stalls at the Trp codons in region 1 because tryptophan is low. Because ribosome is stalled, region 2 is not covered by the ribosome when region 3 is transcribed. 4. When region 3 is transcribed it pairs with region 2. When region 4 is transcribed it cannot pair with 3, because region 3 is already paired with 2; the attenuator never forms and transcript continues
Regulator gene mutation: lacl^s
1. The lacl^s gene produces a super repressor that does not bind lactose 2. The lacl^s gene is trans dominant: the super repressor binds both operators and prevents transcription in the presence and absence of lactose (unlike lacl-, can bind to operator whether lactose is present or not)
When tryptophan is low...
1. The trip repressor is normally inactive 2. Regulatory protein cannot bind to operator and transcription takes place
When tryptophan is high...
1. Tryptophan binds to repressor and makes it active 2. Trp repressor then binds to the operator and shuts transcription off.
Catabolite repression process when glucose is low
1. levels of cAMP are high, cAMP readily binds CAP, and the CAP-cAMP complex binds DNA increasing the efficiency of polymerase binding. 2. The results are high rates of transcription and translation of the structural genes and the production of glucose from lactose.
Antisense RNA for high osmolarity
1. when excel osmolarity is high the micF gene is activated and micF RNA is produced 2. micF RNA pairs with the 5' end of oomph RNA, blocking the ribosom-binding site. No OmpF protein is produced
How many operons are affected by CAP?
20 operons are affected by CAP-- this is how it is regulated
Attenuation results when which regions of the 5′ UTR region pair?
3 and 4
What is the exception with the 5' UTR in the trp operon?
5' UTR Is not normally translated, but in the trap operon it is (exception to the rule)
lac Operon of E. Coli (inducer? does the repression completely shut down)
A negative inducible operon Deals with the metabolism of lactose and regulating the lac operon Inducer: allolactose lacI: repressor encoding gene lacP: operon promoter lacO: operon operator Lactose converted in to other structures via structural genes: lacZ: encoding β-galactosidases lacY: encoding permease lacA: encoding transacetylase The repression of the lac operon never completely shuts down transcription - there are always a few molecules of the structural genes
trp Operon of E. Coli (what kind of operon and what are the structural genes/how many?)
A negative repressible operon- transcription in is normally turned on and must be repressed. - 5: This code for enzyme--> trpE, trpD, trpC, trpB, and trpA—five enzymes together convert chorismate to typtophane.
How does cAMP-CAP affect DNA?
Bends DNA which in turn facilitates RNA polymerase binding to the promoter
Regulatory gene
DNA sequence encoding products that affect that the operon function, but are not part of the operon
Regulatory elements
DNA sequences that are not transcribed but play a role in regulating other nucleotide sequences
Catabolite Repression (What type of control mechanism is this and how does it work?)
E coli. prefer using glucose when available, and repressing the metabolite of other sugars (including lactose) because it takes less energy to metabolize than other sugars. This is a positive control mechanism. The positive effect is activated by catabolite activator protein (CAP). cAMP is binded to CAP, together CAP-cAMP complex binds to a site slightly upstream from the lac gene promoter and increases transcriptional efficiency. The Lac operon is only transcribed when lactose is present and glucose is absent.
Attenuation in the trap Operon (how many many regions are there and of what? What happens when tryptophan is high? When tryptophan is low?)
Four regions of the long 5′ UTR (leader) region of trpE mRNA When tryptophan is high, region 1 binds to region 2, which leads to the binding of region 3 and region 4, terminating transcription prematurely. When tryptophan is low, region 2 binds to region 3, which prevents the binding of region 3 and region 4, and transcription continues.
What does a lacO mutation affect?
LacO mutation affects only genes to which it is physically connected (cis-acting)
What is the effect of high levels of glucose on the lac operon?
Little transcription takes place-- this is independent of lactose
Why Does Attenuation Take Place in the trp Operon?
Repression is never complete; transcription reduced ~ 70-fold, while attenuation reduces another ~ 8-fold to reduce more than 600-fold
Ribsoswitches for low osmolaraity
Riboswitches: molecules influence the formation of secondary structures in mRNA 1. when extracellular osmolarity is low, the oomph mRNA is translated to produce OmpF protein
What is the difference between a structural gene and a regulator gene?
Structural genes encode proteins; regulator genes control the transcription of structural genes.
Negative inducible operons
The control at the operator site is negative (repressor). Molecule binding is to the operator, inhibiting transcription. Such operons are usually off and need to be turned on, so the transcription is inducible. Needs Inducer: small molecule that turns on the transcription. Transcription needs to be turned on because of inducible, but the repressor is turning it off-- so you want to alleviate that and turn on transcription Regulator protein is a REPRESSOR that binds to the operator and prevents transcription of the structural genes (OFF STATE) When the inducer is present it binds to the regulator and makes the regulator unable to bind to the operator. Transcription takes place (ON STATE) The precursor is considered the inducer-- so when you have a lot of the precursor around it will cause it to be made the product-- but once you've gotten rid of it then transcription will stop.
Negative repressible operons
The control at the operator site is negative. But such transcription is usually on and needs to be turned off, so the transcription is repressible. Needs corepressor: a small molecule that binds to the repressor and makes it capable of binding to the operator to turn off transcription. Negative so you're using a repressor-- repressible so you're using the repressor to turn it off Repressor can't normally bind and so transcription occurs (ON STATE) When too much of product is produced-- the product binds to the repressor (this product is called a corepressor) and makes it an active repressor and prevents RNA polymerase from binding
Repressible operons
Transcription is normally on and needs to be turned off.
Inducible operons
Transcription is usually off and needs to be turned on.
How did Jacob and Monod understand the structure and function of the Lac operon?
Used mutations that affected lactose metabolism
Operon
a group of bacterial structural genes that are transcribed together (along with their promoter and additional sequences that control transcription) promoter + additional sequences that control transcription (operator) + structure genes
Cis acting
able to control the expression of genes only when on the same piece of DNA
cAMP (concentration of cAMP)
adenosine-3′, 5′-cyclic monophosphate--a modified nucleotide important for cellular signaling) (The concentration of cAMP is inversely proportional to the level of available glucose. High cAMP=low glucose)
Structural gene mutations
affect the structure of the enzymes (β-galactosidase and permease), but not the regulation of their synthesis - lacZ+lacY− (bacterial chromosome) /lacZ−lacY+ (plasmid) produce fully functional β-galactosidase and permease.
Attenuation
affects the continuation of transcription, not its initiation. This action terminates the transcription before it reaches the structural genes. (NOT initiation it's after everything starts to transcribe)
Operator mutation
altered the sequence of DNA at the operator so that the repressor proteins was no longer able to bind lacO^c c=constitutive
How do attenuation and attenuation and repression response to different singles?
attenuation and repression respond to different signals: repression responds to the cellular levels of tryptophan, whereas attenuation responds to the number of tRNAs charged with tryptophan.
How do amino acids in DNA-binding proteins interact with DNA?
by forming hydrogen bonds with DNA base (hydrogen bonds are relatively weak,t his allows the DNA binding proteins to come off and on easily and compete with other regulatory proteins
In the presence of allolactose, the lac repressor
cannot bind to the operator
Anti-sense RNA
complementary to targeted partial sequenece of mRNA
Constitutive expression
continuously expressed under normal cellular conditions
Regulatory genes
encoding products that interact with other sequences and affect the transcription and translation of these sequences
Structural genes
encoding proteins
Partial diploid
full bacterial chromosome + an extra piece of DNA on F plasmid (have two lac operons now)
Negative control
inhibit gene expression
What is lacl+ over lacl-?
lacI+ is dominant over lacI− and is trans acting. A single copy of lacI+ brings about normal regulation of lac operon.
What produces fully functional β-galactosidase constitutively?
lacI+lacO+Z−/lacI+lacOclacZ+
What produces fully functional B-galatosidase?
lacI+lacZ−/lacI−lacZ+ produce fully functional β-galactosidase.
Regulator gene mutations
lacI− leads to constitutive transcription of three structure genes. (normally lacI = repressor encoding gene)
What is lacO^c in comparison to to lacO+ ?
lacO^c is dominant, which is cis acting
Promoter mutations (lacP-; what fails to produce functional B-galac)
lacP−: cis acting lacI+lacP−lacZ+/lacI+lacP+lacZ− fails to produce functional β-galactosidase.
Ribozymes
mRNA molecules with catalytic activity
Helix-turn-Helix
more common in bacteria (bacterial regulatory proteins ; two alpha helices; major groove (binding site on DNA)
Leucine zipper
more common in eukaryotes
Zinc fingers
more common in eukaryotes Ex: there are numerous Zinc finger DNA-binding proteins that (A) bind to different regulatory proteins and (B) recognize different sets of DNA sequences
When do transcription and translation occur with respect to each other?
nearly simultaneously
LOOK AT WORKED PROBLEM
nope
Where is the lac operator overlaps?
overlaps the 5' end of the lacZ gene
Antiterminator
prevents termination
How are the regions complementary? What happens when tryp is high? low?
region 1 comp to 2 region 2 comp to 3 region 3 comp to 4 1 and 2 and 3 and 4 will bind to each other known as the attenuator when tryptophan is high and transcription terminates 2 and 3 bind and this is the antitermination--> transcript does not terminate
Negative control
regulatory protein is a repressor of transcription
Positive control
regulatory protein is an activator of transcription
If the lac operon is repressed and no permease is being produced, how does lactose get into the cell to inactivate the repressor and turn on transcription? - Furthermore, the inducer is actually allolactose, which must be produced from lactose by β-galactosidase. If β-galactosidase production is repressed, how can lactose metabolism be induced?
repression never completely shuts down. Even with active repressor bound to the operator, there is a low level of transcription and a few molecules of β-galactosidase, permease, and transacetylase are synthesized.
Coordinate induction
simultaneous synthesis of structural proteins stimulated by a specific molecule, the inducer
Positive control
stimulate gene expression (upregulating)
Attenuator
terminates transcription
Positive repressible operon
the regulatory protein is produced in a form that readily binds to DNA, meaning that transcription normally takes place and has to be repressed. A substance must bind to the regulatory protein to render it inactive Once the end product (blue diamond) is in abundance, it binds to the regulatory protein to render it inactive, thus turning off transcription.
How does lacl act?
trans-acting: able to control the expression of genes on other DNA molecules (chromosome and F plasmid)
Positive inducible operon
transcription is normally turned off because the regulator protein (an activator) is produced in an inactive form. Transcription takes place when an inducer is attached to the regulatory protein, rendering the regulator active. (inactive activator needs substrate to activate the activator and bind to the DNA and activate transcription) When the enzyme is low (blue diamond), the precursor (purple circle) is high, thus turning transcription on to make the enzyme. The opposite is true, so the system/balance is economical.
What is within region 1?
two trp codons within region one
Motif
within the binding domain, a simple structure that fits into the major groove of the DNA Distinctive types of DNA-binding proteins based on the motif (that is, any given DNA-binding protein may have a particular type of motif that recognizes and binds a specific DNA sequence)
Domains
~ 60 - 90 amino acids, responsible for binding to DNA, forming hydrogen bonds with DNA (usually just part of the protein called the DNA-binding domain). These proteins can have different types of domains that bind other regulatory proteins, but typically only have 1 DNA-binding domain.
