Question

PLEASE help answer all of question 18 a-e

18. What would the lac operon look like in a eukaryote? List two possible changes in chromatin. Explain them to each other. a. b. Transcriptional control. Draw how a single gene (Lacz) would be transcribed and regulated c. If they are each in different chromosomes, draw how their expression would be coordinated Processing control. Pick one approach and explain to your partners d. Translational regulation. Pick one approach and explain to your partners e.

Answer 1

Answer a -  

Answer - chromatin

Chromatin is a complex of DNA and proteins that forms chromosomes within the nucleus of eukaryotic cells. Nuclear DNA does not appear in free linear strands; it is highly condensed and wrapped around nuclear proteins in order to fit inside the nucleus.

Chromatin exists in two forms. One form, called euchromatin, is less condensed and can be transcribed. The second form, called heterochromatin, is highly condensed and is typically not transcribed.

Under the microscope in its extended form, chromatin looks like beads on a string. The beads are called nucleosomes. Each nucleosome is composed of DNA wrapped around eight proteins called histones. The nucleosomes are then wrapped into a 30 nm spiral called a solenoid, where additional histone proteins support the chromatin structure. During cell division, the structure of the chromatin and chromosomes are visible under a light microscope, and they change in shape as the DNA is duplicated and separated into two cells.

Chromatin Remodeling

Various molecules called chromatin remodelers provide the mechanism for modifying chromatin and allowing transcription signals to reach their destinations on the DNA strand. Understanding the nature and processes of these cellular construction workers remains an active area of discovery in genetic research.

Currently, investigators know that chromatin remodelers are large, multiprotein complexes that use the energy of ATP hydrolysis to mobilize and restructure nucleosomes. Recall that nucleosomes wrap 146 base pairs of DNA in approximately 1.7 turns around a histone-octamer disk, and the DNA inside each nucleosome is generally inaccessible to DNA-binding factors. Remodelers are thus necessary to provide access to the underlying DNA to enable transcription, chromatin assembly, DNA repair, and other processes. Just how remodelers convert the energy of ATP hydrolysis into mechanical force to mobilize the nucleosome, and how different remodeler complexes select which nucleosomes to move and restructure, remains unknown, however.

Remodelers are partitioned into five families, each with specialized biological roles. Nonetheless, all remodelers contain a subunit with a conserved ATPase domain. In addition to the conserved ATPase, each remodeler complex also possesses unique proteins that specialize it for its unique biological role. However, because all remodelers move nucleosomes and all such movement is ATP dependent, mobilization is most likely a property of the conserved ATPase subunit.

The ATPase domains of remodelers are similar in sequence and structure to known DNA-translocating proteins in viruses and bacteria. Recent evidence from the SWI/SNF and ISWI remodeler families has also revealed that remodeler ATPases are directional DNA translocases that are capable of the directional pumping of DNA. But how is this property applied to nucleosomes? It seems that the ATPase binds approximately 40 base pairs inside the nucleosome, from which location it pumps DNA around the histone-octamer surface. This enables the movement of the nucleosome along the DNA, thus permitting the exposure of the DNA to regulatory factors.

The additional domains and proteins that are attached to the ATPase are important for nucleosome selection, and they also help regulate ATPase activity. These attendant proteins bind to histones and nucleosomal DNA, and their binding to these molecules is affected by the histone modification state. The modification state helps determine whether the nucleosome is an appropriate substrate for a remodeler complex .

Signaling Function of Remodeled Chromatin

Histone modification can open chromatin, thus permitting selective binding of transcription factors that, in turn, recruit RNA polymerase II (Turner, 2005). Varying levels and types of histone modifications have been shown to correlate with levels of chromatin activation. For example, one group of researchers used antibody-based immunoprecipitation studies to determine that acetylation of histone H3 and methylation at lysine residue K4 appeared to coincide with each other. They also coincided during transcriptional activation in chicken embryos, while methylation at lysine residue K9 marked inactive chromatin.

DNA Methylation

Another means by which transcription is controlled is through methylation of the DNA strand itself. Not to be confused with histone methylation, methylation of the DNA strand involves cytosine bases of eukaryotic DNA being converted to 5-methylcytosine, resulting in the repression of transcription, particularly in vertebrates and plants. The altered cytosine residues are usually immediately adjacent to a guanine nucleotide, resulting in two methylated cytosine residues set diagonally to one another on opposing DNA strands

Heavily methylated regions of DNA with elevated concentrations of these so-called CpG groups are often found near transcription start sites. In an interestingly coordinated process, proteins that bind to methylated DNA also form complexes with proteins involved in deacetylation of histones. Therefore, when the DNA is in a methylated state, nearby histones are deacetylated, resulting in compact, semipermanently silent chromatin. Likewise, demethylated DNA does not draw deacetylating enzymes to the histones, but it often attracts histone acetyltransferases, allowing histones to remain acetylated and promoting transcription.

Methylated De novo methylation (DNMT3A, DNMT3B) Unmethylated ATCGAATGCTGCGGA ATCGAATGCTGCGGA TAGCTTACGACGCCT TAGCTTACGACGCCT Epigenic reprogramming Sinng complex histone deacetylation, histone H3K9 methylation, HP1 binding) Restored methylation Silenced ATCCAATGCTGCGGA ATCGAATGCTGCGGA TAGCTTACGACGCCT Hemi-methylated Maintenance methylation DNMT1) DNA replication Nature Reviews Cancer

Answer b -  

Prokaryotic Gene Regulation: Regulation of the lac operon (dual control: repression and promotion)

Example of prokaryotic gene control: the lac operon.
The best example of genetic control is the well studied system of milk sugar (lactose) inducible catabolism in the human symbiote, Escherichia coli.
The lac operon includes 3 structural genes (lacZ, lacY and lacA) that are transcribed in unison.
Located near the lac operon, is the lacI gene regulates the operon by producing the lac repressor protein.
Both the regulatory gene and the lac operon itself contain...
1) promoters (P_l and P_lac) at which RNA polymerase binds and
2) terminators at which transcription halts.
P_lac overlaps with the operator site (O) to which the active form of the repressor protein binds.
The operon is transcribed into a single long molecule of mRNA that codes for all three polypeptides.

Transcription of the lac operon is down-regulated through the binding of the lac repressor to the operator.
In the absence of lactose, the repressor remains bound to the operator and preventing access of the RNA polymerase to the promoter.
Transcription is blocked and the operon is repressed.
In the presence of lactose, the repressor is inactivated form and does not bind to the operator.
Thus the RNA polymerase may bind to the promoter and transcribe the structural genes into a single cistronic mRNA.
The isomeric form of lactose that binds to the repressor is allolactose.
The lac repressor is an allosteric protein capable of reversible conversion between two alternative forms.
In the absence of the effector allolactose, the protein is in the form that binds to the lac operator.
In the effector�s presence, the repressor mostly exists in the alternative and inactive state.

Transcription of the lac operon is up-regulated through the binding of the cAMP Receptor Protein (CRP) complex to the promoter.
The cAMP Receptor Protein (CRP) is an allosteric protein that is inactive in the free form but is activated by binding to cAMP.
The CRP-cAMP complex binds the promoter of inducible operons, increasing the affinity of the promoter for RNA polymerase to stimulate transcription.
The effect of active CRP on the lac operon.
1) The CRP-cAMP complex binds to the CRP recognition site near the promoter region, thereby
2) making the promoter more readily bound by RNA polymerase.
3) RNA polymerase binds to the promoter and transcribes the operon.

Prokaryotic Gene Regulation: Regulation of the trp operon (a "riboswitch")

An example of genetic control in prokaryotes: the trp operon
The trp operon includes five structural genes (trpE, trpD, trpC, trpB, and trpA) as well as promoter (P_trp), operator (O), and leader (L) sequences.
The structural genes are transcribed and regulated as a unit.
The repressor protein, encoded by the trpR gene is inactive (cannot recognize the operator site) in the free form when tryptophan is not abundant.
The polycistronic mRNA encodes for the enzymes of the tryptophan biosynthetic pathway.
When complexed with tryptophan, the repressor is active and binds tightly to the operator, blocking access of RNA polymerase to the promoter and keeping the operon repressed.

In prokaryotes, no nuclear membrane separates transcription and translation and the ribosomes will bind the nascent message soon after it emerges from the RNA polymerase.
The close linkage of the processes can lead to interdependent control mechanisms such as the attenuation controlled by the trp leader sequence.
The transcript of the trp operon includes 162 nucleotides upstream of the initiation codon for trpE (the first structural gene).
This leader mRNA includes a section encoding a leader peptide (or sensor) of 14 amino acids.
In short, if tryptophan is present (in moderate amounts), the sensor peptide is easily made and the long trp operon mRNA is NOT completed.
If tryptophan is scarce, the leader peptide is not easily made and the full operon is transcribed then translated into tryptophan synthetic enzymes.
Two adjacent tryptophan (trp) codons within the leader mRNA sequence are essential in the operon's regulation.
The leader mRNA contains four regions capable of base pairing in various combinations to form hairpin structures.
Attenuation depends upon the ability of regions 1 and 2 and regions 3 and 4 of the trp leader sequence to base pair and form hairpin secondary structures.
A part of the leader mRNA containing regions 3 and 4 and a string of eight U's is called the attenuator.
The region 3+4 hairpin structure acts as a transcription termination signal; as soon as it forms, the RNA and the RNA polymerase are released from the DNA.
During periods of tryptophan scarcity, a ribosome translating the coding sequence for the leader peptide may stall when it encounters the two tryptophan (trp) codons because of the shortage of tryptophan-carrying tRNA molecules.
Because a stalled ribosome at this site blocks region 1, a region 1+2 hairpin cannot form and an alternative, region 2+3 hairpin is formed instead.
The region 2+3 base pairing prevents formation of the region 3+4 transcription termination hairpin and therefore RNA polymerase can move on to transcribe the entire operon to produce enzymes that will synthesize tryptophan.
When tryptophan is readily available, a ribosome can complete translation of the leader peptide without stalling.
As it pauses at the stop codon, it blocks region 2, preventing it from base pairing.
As a result, the region 3+4 structure forms and terminates transcription near the end of the leader sequence and the structural genes of the operon are not transcribed (nor translated).
This is example of a "riboswitch", a mechanism which can control transcription and translation through intereactions of molecules withe an mRNA.

operon structural genes promoter operator regulatory gene DNA transcription mRNA translation protein protein protn protein
AAnswer c-
ATP high glucose active inactive adenyl cyclase adenyl low glucose cyclase inactive catabolite activator protein catabolite activator protein binds cAMP, changes conformation, binds CAP site activates transcription of lac operon lac Z lacl CAP p lac Y lac A ac O lac operorn transcribed at lac I gene transcribed and translated into lac repressor very high level repressor binds lactose, changes conformation, can't bind to operator lactose