What are histones and chromatin? Describe DNA packing in a ...
What are histones and chromatin? Describe DNA packing in a nucleosome. Eukaryotic chromosomes have linear DNA molecules associated with a large amount of protein. Chromatin is a complex of DNA and protein and is found in the nucleus of eukaryotic cells. Chromatin causes condensation and organization of DNA The protein:DNA mass ratio is 2:1 and the histone:DNA mass ratio is 1:1. Chromatin includes several levels of condensation including 10 nm fibres with nucleosomes (bead on a string-like configuration) which offer 7x compaction. The nucleosomes are repeat units of chromatin. This is the primary structural unit and is controlled by histones. Histones are the units that DNA winds around to the form the "beads". Histone structure: Has a 3-helix core domain and forms a handshake like arrangement. The tails are N-terminal or C-terminal that protrude from the nucleosome through minor groove channels. They are in the ideal location for covalent modifications. It is an octamer made of up H3H4 tetramer and 2 H2A-H2B dimers. Formation of the 30 nm fibers which offer 40X compaction provide the second level of compaction. This is achieved through histone tail-mediated nucleosome-nucleosome interactions. Tertiary structures are formed by tail-mediated association of individual fibres. What positions nucleosomes and what are the functions of chromatin remodelling ATPases? Nucleosomes are positioned by transcription factors, general transcriptional machinery and chromatin remodelling ATPases. The chromatin remodelling ATPases have several functions: transcription, repression, DNA methylation, exchange, repair and recombination. They bind nucleosomes, are DNA-dependant ATPases, recognize histone modifications, can be regulated and interact with other proteins. What are proteosomes and what is their importance in ubiquination? Proteosomes are giant protein complexes that bind protein molecules and degrade them. The protein to be degraded is ubiquitinated and sent to the proteosome with this tag to be degraded. The proteosome and the ubiquitin are recycled and the protein ends up in fragments. What is RNA interference? Explain roles of siRNA and miRNA mediated interference mechanisms. Inhibition of gene expression by RNA molecules is called RNA interference (RNAi). RNAi is caused by small interfering RNAs (siRNAs). siRNAs play a role in heterochromatin formation and can block large regions of a chromosome. Small RNAs can stop transcription of a specific gene. MicroRNAs or miRNAs are small single stranded RNA molecules that bind to mRNA and they can degrade the piece of RNA or stop it's translation.
Give one example each of a positive and negative regulated operon in prokaryotes. Operon: A cluster of functionally related genes can be under coordinated control by a single on-off "switch". The regulatory "switch" is a segment of DNA called an operator usually positioned within the promoter. An operon consists of the entire stretch of DNA that includes the operator, the promoter and the genes that they control. The operon can be switched off a protein called a repressor. The repressor prevents gene transcription by binding to the operator and blocking RNA polymerase. The repressor is the product of a separate regulatory gene. The repressor can sometimes work with a co-repressor. (ex. E. coli can synthesize the amino acid tryptophan, tryptophan binds to repressor when produced). Negatively regulated operon (operons are switched off by the active form of the repressor) : lac operon. It is an inducible operon and contains genes that code for enzymes used in the hydrolysis and metabolism of lactose. The lac repressor is usually active and switches the operon off but a molecular called an inducer can inactivate the repressor and turn the lac operon on. Positive gene regulation: Operons are subject to positive control through a stimulatory protein, such as a catabolite activator protein (CAP), which is an activator of transcription. Ex. when glucose is scarce, CAP is activated by binding with cAMP. Activated CAP attaches to the promoter of the lac operon and increases affinity to RNA polymerase, accelerating transcription. When glucose levels increase, CAP detaches from the lac operon and transcription returns to a normal rate. Is it guaranteed that once mRNA is expressed that the encoded gene product will also be expressed? Why or why not? No, initiation of translation of selected mRNAs can be blocked by regulatory proteins that bind to sequences or structures of the mRNA. After translation, various types of protein processing including cleavage and addition of chemical groups are subject to control. How does the presence of glucose in the medium in which E. Coli is grown affect the synthesis of E.Coli's enzymes involved in the utilization of lactose? What happens at the molecular level to explain this effect? Levels of glucose activate or deactivate lactose metabolism genes in E.Coli. When glucose is scarce, CAP is activated by binding with cAMP. Activated CAP attaches to the promoter of the lac operon and increases affinity to RNA polymerase, accelerating transcription. When glucose levels increase, CAP detaches from the lac operon and transcription returns to a normal rate Name a compound that only acts as an inducer of the lac operon and explain why it cannot be a substrate. Name a compound that is a substrate for bgalactosidase and explain why it cannot act as an inducer.
Describe transcriptional attenuation. Why hasn't this regulatory mechanism been described in eukaryotic cells?
An example is the trp gene in bacteria. When there is a high level of tryptophan in the region, it is inefficient for the bacterium to synthesise more. When the RNA polymerasebinds and transcribes the trp gene, the ribosome will start translating. (This differs from eukaryotic cells, where RNA must exit the nucleus before translation starts.) The attenuator sequence, which is located between the mRNA leader sequence (5' UTR) and trp operon gene sequence, contains four domains, where domain 3 can pair with domain 2 or domain 4. The attenuator sequence at domain 1 contains instruction for peptide synthesis that requires tryptophans. A high level of tryptophan will permit ribosomes to translate the attenuator sequence domains 1 and 2, allowing domains 3 and 4 to form a hairpin structure, which results in termination of transcription of the trp operon. Since the protein coding genes are not transcribed due to rho independent termination, no tryptophan is synthesised. Compare and Contrast repression in the lac and trp operons. A repressible operon is one that is usually on; binding of a repressor to the operator shuts off transcription. The trp operon is a repressible operon. It is usually on and the genes for tryptophan synthesis are transcribed. When tryptophan is present, it binds to the trp repressor protein which turns the operon off. The repressor is active only in the presence of its corepressor protein which turns the operon off. An inducible operon is one that is usually off; a molecule called an inducer inactivates the repressor and turns on transcription. The lac operon is an inducible operon and contains genes that code for enzymes use in the hydrolysis and metabolism of lactose. The lac repressor is usually active and switches the lac operon off. A molecule called an inducer inactivates the repressor to turn the lac operon on.
Give one example each of a positive and negative regulated operon in prokaryotes. Operon: A cluster of functionally related genes can be under coordinated control by a single on-off "switch". The regulatory "switch" is a segment of DNA called an operator usually positioned within the promoter. An operon consists of the entire stretch of DNA that includes the operator, the promoter and the genes that they control. The operon can be switched off a protein called a repressor. The repressor prevents gene transcription by binding to the operator and blocking RNA polymerase. The repressor is the product of a separate regulatory gene. The repressor can sometimes work with a co-repressor. (ex. E. coli can synthesize the amino acid tryptophan, tryptophan binds to repressor when produced). Negatively regulated operon (operons are switched off by the active form of the repressor) : lac operon. It is an inducible operon and contains genes that code for enzymes used in the hydrolysis and metabolism of lactose. The lac repressor is usually active and switches the operon off but a molecular called an inducer can inactivate the repressor and turn the lac operon on. Positive gene regulation: Operons are subject to positive control through a stimulatory protein, such as a catabolite activator protein (CAP), which is an activator of transcription. Ex. when glucose is scarce, CAP is activated by binding with cAMP. Activated CAP attaches to the promoter of the lac operon and increases affinity to RNA polymerase, accelerating transcription. When glucose levels increase, CAP detaches from the lac operon and transcription returns to a normal rate. Is it guaranteed that once mRNA is expressed that the encoded gene product will also be expressed? Why or why not? No, initiation of translation of selected mRNAs can be blocked by regulatory proteins that bind to sequences or structures of the mRNA. After translation, various types of protein processing including cleavage and addition of chemical groups are subject to control. How does the presence of glucose in the medium in which E. Coli is grown affect the synthesis of E.Coli's enzymes involved in the utilization of lactose? What happens at the molecular level to explain this effect? Levels of glucose activate or deactivate lactose metabolism genes in E.Coli. When glucose is scarce, CAP is activated by binding with cAMP. Activated CAP attaches to the promoter of the lac operon and increases affinity to RNA polymerase, accelerating transcription. When glucose levels increase, CAP detaches from the lac operon and transcription returns to a normal rate Name a compound that only acts as an inducer of the lac operon and explain why it cannot be a substrate. Name a compound that is a substrate for bgalactosidase and explain why it cannot act as an inducer.
Describe transcriptional attenuation. Why hasn't this regulatory mechanism been described in eukaryotic cells?
An example is the trp gene in bacteria. When there is a high level of tryptophan in the region, it is inefficient for the bacterium to synthesise more. When the RNA polymerasebinds and transcribes the trp gene, the ribosome will start translating. (This differs from eukaryotic cells, where RNA must exit the nucleus before translation starts.) The attenuator sequence, which is located between the mRNA leader sequence (5' UTR) and trp operon gene sequence, contains four domains, where domain 3 can pair with domain 2 or domain 4. The attenuator sequence at domain 1 contains instruction for peptide synthesis that requires tryptophans. A high level of tryptophan will permit ribosomes to translate the attenuator sequence domains 1 and 2, allowing domains 3 and 4 to form a hairpin structure, which results in termination of transcription of the trp operon. Since the protein coding genes are not transcribed due to rho independent termination, no tryptophan is synthesised. Compare and Contrast repression in the lac and trp operons. A repressible operon is one that is usually on; binding of a repressor to the operator shuts off transcription. The trp operon is a repressible operon. It is usually on and the genes for tryptophan synthesis are transcribed. When tryptophan is present, it binds to the trp repressor protein which turns the operon off. The repressor is active only in the presence of its corepressor protein which turns the operon off. An inducible operon is one that is usually off; a molecule called an inducer inactivates the repressor and turns on transcription. The lac operon is an inducible operon and contains genes that code for enzymes use in the hydrolysis and metabolism of lactose. The lac repressor is usually active and switches the lac operon off. A molecule called an inducer inactivates the repressor to turn the lac operon on.
