L. 20; Gene Regulation (Ch.15)

four possible combinations of regulation

1. negative inducible. example: lac repressor + lactose. 2. positive inducible. example CAP + cAMP. 3. negative repressible. example trp repressor and tryptophan. 4. positive repressible.

alternate splicing: Male vs Female

5' splice site: active Sxl protein is only made in female fruit flies -because the third exon contains a stop codon. 3' splice site: active Tra, transformer, protein is also only made in females because of the presence of Sxl protein.

several different binding sites may be found in eukaryotic genes

MRE - multiple response element. TRE - transcription response element. GRE - glucocorticoid response element. Enhancer - sequences that stimulates high levels of transcription, sometimes at a long distance from the promoter.

attenuation when trp levels are high

a hair pin loop forms between repeat sequences 1 and 2 and then a transcription termination hairpin loop also forms between repeats 3 and 4 followed by UUUUUU in the 5'-UTR region of the mRNA. this causes the RNA polymerase to fall of the DNA after repeat 4 while still in the 5'-UTR

DNA methylation in eukaryotes

a mechanism to inhibit transcription. on cytosine base. CpG islands are areas of DNA with many of these CG sites where transcription can be repressed by methylation.

steroid hormones

act as effector molecules to stimulate transcription. the steroid hormone enters its target cell and combines with a receptor protein. the hormone/protein complex binds to a hormone response element in the DNA. the bound complex stimulates transcription. transcript is process and transported into the cytoplasm. the mRNA is translated into protein.

histone modifications and the histone code

addition of acetyl groups to the histone protein tails cause the DNA-histone interaction to relax. transcription factors can then bind to the DNA. phosphorylation and methylation of histone tails can also occur to influence gene expression. the sum of these modifications is called the histone code.

additional level where gene expression can regulated in eukaryotes

alteration of chromatin structure.

trp operon is also regulated by another mechanism...

attenuation. this mechanism involves the coupling of transcription to translation.

CAP and cAMP

catabolite activator protein, a regulatory protein. CAP binds to form CAP/cAMP complex.

gene regulation by RNA interference

double strand RNA present in a cell can be cleaved into small pieces of 21-25 bp in length by Dicer RNase. these RNA pieces called silencing RNAs (siRNAs) or microRNAs (miRNAs) bind to a protein complex called RISC (RNA-induced silencing complex)/ RISC-siRNAs binds to complementary mRNA to cause mRNA degradation. RISC-miRNAs bind complementary mRNAs to cause inhibition of protein translation.

enhancers, silencers, and insulators

enhancers can cause significant increase in transcription levels, often working at a distance from the promoter. silencers are similar to enhancers, but they repress transcription. insulators can block the effect of an enhancer or silencer element.

eukaryotic gene regulation

eukaryotes can be multicellular. different tissue types, different developmental stages. genes are regulated spatially. different gene expression in different tissue types. genes are regulated temporally. different gene expression at different times.

chromatin structure

eukaryotic DNA is wound up on nucleosomes. it must be un-packaged before transcription can occur.

temporal regulation of alpha and beta globin genes

globin genes are a multigene family. at any times in development, globin consists of 2 alpha and 2 beta subunits + heme. but different alpha and beta genes are used which encode slightly different polypeptides. the psi genes are pseudo genes - ancestral forms of globin genes that are no longer functional.

CAP/cAMP complex

is a positive inducible regulatory mechanism of the lac operon. CAP = positive regulator (activator) cAMP = effector molecule = inducer.

the trp operon

is for the biosynthesis of trptophan. converts chorismate to tryptophan. a negative repressible operon: negative = repressor. repressible = normally on, uses co-repressor. product tryptophan is a co-repressor which binds repressor to turn off operon expression.

negative repressible operons vs negative inducible operons

negative repressible operons, trp, are used for biosynthesis. negative inducible operons, lac, are used for degradation.

no glucose with presence of lactose

no glucose results in the making of cAMP. cAMP is high so it forms the CAP/cAMP complex. the complex binds to the promoter and enhances binding of RNA polymerase. presence of lactose binds to the lac repressor so the lac repressor is not bound to the operator. = very high levels of lac mRNA.

chromatin remodeling

nucleosome remodeling factors can alter the positioning of nucleosomes to allow transcription of genes to occur.

spatial regulation of tubulin genes

one variation of an alpha-tubulin gene in mustard plant is only expressed in the pollen grains. this gene is fused to the e. coli lac z gene to make its expression visible (after adding lactose-conjugated blue dye)

glucose levels

regulate the levels of cAMP. high glucose inactivates adenyl cyclase so ATP makes no cAMP. low glucose does not inactivate adenyl cyclase, ATP makes cAMP.

tissue-specific enhancers

spatial regulation. - Drosophila yellow gene - transcription of the gene is activated in different tissues through separate enhancer elements.

another form of gene regulation: alternate splicing of pre-mRNA

splicing at different sites leads to different protein.

When glucose is present in E. coli

the lac operon is not needed. induction of the lac operon is actively repressed even in the presence of lactose.

promoter-operator sequence of the lac operon

the lac promoter has a CAP/cAMP binding site upstream of the RNA polymerase binding site. operator is downstream from the binding site. CAP/cAMP binding enhances binding of RNA polymerase here. the binding of the cAMP/CAP complex to DNA produces a sharp bend in DNA that activates transcription.

mRNA stability is a mechanism of gene regulation

the longer the mRNA is available to be translated, the more protein that can be made. different mRNAs have different lifespans in the cytoplasm. stability of the mRNA is affected by the: -length of the poly-a tail. - sequences of the 3' untranslated region. - presence/absence of RNA binding proteins.

transcription of eukaryotic genes:

the naked DNA is not transcribed. DNA is in nucleosomes. ground states is usually OFF. binding of transcription factors is required for transcription to occur, so mostly transcription regulation is positive control.

transcription of prokaryotic genes:

the naked DNA is transcribed. ground state is usually ON. transcription levels can be increased through the interactions of activator proteins with RNA polymerase. however, repressor are more common. mostly negative control.

high glucose and lactose

the presence of glucose make the cAMP concentration low, too low to form the CAP/cAMP complex. CAP does not bind. presence of lactose binds to the lac repressor so the lac repressor is not bound to the operator. = very low level of lac mRNA

when tryptophan is low:

the repressor is inactive and the operon is transcribed.

attenuation when trp levels are low

the ribosome stalls on two trp codons in region 1 of the mRNA waiting for trp-charged tRNA. this prevents the hairpins forming between repeats 1 and 2. instead a hairpin forms between repeats 2 and 3. this prevents the transcription termination hairpin from forming between repeats 3 and 4 and transcription continues normally.

when tryptophan is high

tryptophan is the co-repressor, when it is high it binds to the repressor which binds DNA to block transcription of the operon.

high glucose, no lactose

when glucose is present cAMP is too low to form the CAP/cAMP complex. no lactose results in no allolactose binding to the lac repressor so the lac repressor is bound to the operator. = no lac mRNA.