genetics chapter 17
Promoters contain proximal-promoter elements
-Located upstream of TATA and BRE motifs -Enhance levels of basal transcription -Ex: CAAT and GC boxes -these are recognized by transcription factors that stimulate transcription at promoter. -These are elements that modulate the efficiency of basal levels of transcription. -act long with the core promoter elements to increases the levels of basal transcription. -CAAT box is located about 70-80bp upstream from start site. When CAAT boxes are present they are critical to the promoter's ability to initiate transcription. Mutations on either side of the box have no impact, but mutation within the CAAT box dramatically lowers the rate of transcription. -Another element is the GC box located 100bp upstream.
Posttranscriptional regulation
-Plays an equal, if not more significant role compared to transcriptional control (a major type of regulation in eukaryotes) -Mechanisms of posttranscriptional gene regulation: -Control of polyadenylation -Alternative splicing -mRNA stability -Translation -RNA silencing.
RNA-induced gene silencing
-RNA interference (RNAi): -Post transcriptional regulation -short RNA molecules regulate gene expression in cytoplasm of plants, animals, and fungi -repress translation and trigger mRNA degration -RNA-induced transcription silencing: chromatin modifications to repress transcription -These phenomena are known as RNA induced gene silencing
Alternative splicing
*CT mRNA/ CGRP mRNA example* -Alternative splicing can generate different forms of mRNA from identical pre-mRNA molecules so that expression of one gene can give rise to a number of proteins with similar or different functions. -Changes in splicing pattern can have many different effects on the translated protein. -Primary transcript contains six exons and it can be spliced into two different mRNAs, both of which contain the first three exons but differ in the final ones. -in CT mRNA (thyroid hormones), it contains exons 1,2,3,4 and the exon 4 polyadenylation signal is used to process the mRNA and add the poly A tail. This mRNA is translated into the calcitonin peptide. -In CGRP mRNA (neuronal cells, it contains exons 1,2,3,5,6 but not exon 4. Exon 6 polyadenylation site gets a poly A tail added and then translated into CGRP peptide. *Dscam Gene example (drosphilla)* - Neurons have cellular processes called axons that form connections with other nerve cells. -Dscam gene encodes a protein that guides axon growth, ensuring the neurons are correctly wired together. -pre mRNA consist of exons 4,6,9,17 that each consist of an array of possible alternatives. they are spliced into the mature mRNA in an exclusive fashion, so that each exon is represented by no more than one of it's possible alternatives. -you can expand the potential of the genomic info to create more variety of proteins.
Great diversity exists in eukaryotic promoters, in structure and functions
-A promoter is a region of DNA the recognizes the transcription machinery and binds one or more proteins that regulate transcription initiation. Promoters are necessary in order for transcription to be initiated accurately and at a basal level. -Promoter elements are short nucleotide sequences that bind specific regulatory factors. -Focuses promoters specify transcription initiation at a single nucleotide (transcription start site). -Dispersed promoters direct initiation from a number of weak transcription start sites located over a 50-100 nucleotide region. -Focused promoters are associated with genes whose transcription levels are highly regulated where dispersed promoters are associated with genes that are transcribed constitutively.
Gene expression influenced by chromatin modifications
-DNA methylation at CG dinucleotides (c-methylation) inhibits transcription -Eukaryotic DNA is combined with histones and non-histone proteins to form chromatin -Compact chromatin structure inhibits transcription, replication and DNA repair -Histone modifications: covalent bonding of functional groups onto N-terminal tails of histone proteins. Most common functional groups are acetyl, methyl, phosphate. -HAT= Histone Acetyl Transferase Enzymes -HDAC= Histone Deacetylase enzymes -The more methylation, the greater the inhibition of transcription. -You have to be able to free up DNA for proteins to bind. -DNA methylation occurs at position 5 of cytosine, causing the methyl group to protrude into the major groove of the DNA helix. There is methylation of bases and sugars. -Methylation represses transcription by inhibiting the binding of transcription factors to DNA. -Acetylation decreases the positive charge on histones, resulting in a reduced affinity of the histone for DNA. In turn, this may assist in the formation of open chromatin conformations which would allow the binding of transcription regulatory proteins to DNA. -Histone acetylation is catalyzed by HATS. They are recruited to genes by the presence of certain transcription activator proteins that bind to transcription regulatory regions. sometimes transcription activator proteins THEMSELVES have HAT activity. -HDAC removes acetate groups from histone tails. They can be recruited to genes by presence of certain repressor proteins on regulatory regions. -You need to remove regions of histones and you can do that by modifying them. The amino terminal regions have tails which are positively charged amino acids that bind and strengthen DNA. You can weaken the interactions of tail and free up DNA for transcription factors to bind. -HAT= add acetyl to histones to stimulate transcritpion -HDAC= remove acetyl groups and stimulate repression.
Enhanceosome model
-Explains how transcription activators and repressors bring about changes to RNAP II transcription -Involves the formation of DNA loops that bring distant enhancer or silencer elements into close physical contact with the promoter regions of the genes that they regulate. -DNA looping may deliver activators, repressors, and general transcription factors to the vicinity of the promoters that must be activated or repressed. -Enhancer and silencer elements act as donors that increase the concentrations of important regulatory proteins at gene promoters by enhancing the rate of the PIC (pre initiation complex) assembly or stability, or accelerating the release of RNAP II from the promoter. -Transcription activators bound at enhancers may stimulate the rate of transcription initiation. -In order for this to happen, activators interact with co activators that form the enchanceosome complex. -Enhanceosomes may directly contact the PIC through subunits of the mediator and TFIID (TAF+TBP). -repressors bound at silencer elements may decrease the rate of PIC assembly and the release of RNAP II. -DNA looping may result in chromosome alterations that either stimulate or repress transcription of target genes.
RNA interference
-First, siRNA or miRNA molecules associtate with an ezyme complex called the RISC complex. Within the RISC, the short double stranded RNA is denatured and the antisense strand is degraded. Then RNA/RISC complex becomes a functional and highly specific agent of RNAi, seeking out mRNA molecules that are complementary to the antisense RNA contained in RISC. -At this point, it can take two pathways: 1.) If the antisense RNA in the RISC is perfectly complementary to the mRNA, the RISC will cleaved the mRNA. The cleaved mRNA is then degraded by ribonucleases. 2.) if the antisense RNA is not exactly complementary to the mRNA, the RISC complex stays bound to the mRNA, interfering with the ability of ribosomes to translate the mRNA. -miRNA or siRNa therefore either repress translation or trigger mRNA degration. -siRNAs and miRNAs can also repress transcription of specific genes and larger regions of the genome, by associating with the complex RITS. The antisense RNA within the RITS targets the RITS complex to specific gene promoters or larger regions of chromatin. RITS then recruits chromatin modifying enzymes to these regions, and they methylate histones and DNA resulting in heterochromatin formation and transcriptional silencing. They can also repress in a direct way, by targeting transcription factors mRNA to reduce the levels of transcription factors of genes who depend on these factors, and they are repressed.
Core promoter
-Made up of DNA sequence elements including: -Initiator (Inr) -TATA box -TFIIB recognition element (BRE) -Downstream promoter element (DPE) -Motif ten element (MTE) -These elements help bind transcription factors and help recruit RNA polymerase. -Promoters may lack the TATA box but the MTE and DPE can substitute for the function of the TATA box. -The more of these sites you have present the better the efficiency of binding -The core promoter dertemines the accurate initiation of transcription of RNA pol II. -Inr element is the transcription start site.
hMTIIA (human metallothionein IIA gene)
-Protein product binds to heavy metals such as zinc and cadmium, to protect cells from toxic effects of high levels of these metals. -Example of how a gene can be transcriptionally regulated due to interplay of promoters, enhancer elements, and transcription factors that bind to them. -Also protects cells from the effects of oxidative stress. -Expressed at low levels in all cells but is transcribed at high levels when cells are exposed to metals or steroid hormones.
Product of hMTIIA
-Protein that binds heavy metals and protects cells from toxic effects -Protects cells from oxidative stress -Expressed in low levels in all cells -Transcribed at high levels when exposed to heavy metals -SP1 factor stimulates transcription at low levels in most cells. -BLE= basal element (enhancer element) -ARE=AP factor response element (enhancer element). -These cis-elements bind the activator proteins 1,2,4 which are present at various levels in different cell types and can be activated in response to extracellular growth signals. BLE contains overlapping binding sites for AP1 and AP4 factors which provides some selectivity in how the factors stimulate transcription when bound to BLE in different cell types. -High levels of transcription are stimulated by the presence of enhancers MRE (metal response element) and GRE (glucocorticoid response element). -Metal inducible transcription factor (MTF1) binds MRE in response to presence of heavy metals. -Glutocorcorticoid receptor protein binds to GRE only when the receptor protein is in a complex with the glucocorticoid steroid hormone. -Transcription of the hMTIIA gene can be repressed by repressor protein PZ120, which binds over the transcription start region.
Nucloesome Remodeling complexes
-eg. SWI/SNF complex -It has an DNA-stimulated ATPase activity. -Uses ATP energy to destabilize histone-DNA complexes. -You need to be able to clear up some space to make DNA accessible to transcription factors. -ATP hydrolysis to move and rearrange nucelosomes along DNA. -repositioned nucleosomes make regions of chromosome accessible to transcription regulatory proteins, such as transcription activators and RNAP II. -one way SWI/SNF acts is by loosening the attachment between histones and DNA, resulting in the nucleosome sliding along the DNA and exposing regulatory regions. -Another way is to loosen the DNA strand from the nucelosome core -Another way is to cause reorganization of the internal nucelosome components. Can cause histone dimer exchange. there are other variants that can be exchanged and they replace that location and bind less tightly, which causes movement of nucelosomes and stimulate transcription. -In all cases the DNA is left exposed to association with transcription factors and RNA polymerase. -Chromatin remodeling complexes can be recruited to DNA by transcription activator proteins that are bound to specific regions of DNA. Their interactions may also be affected by the presence or absence of histone modifications.
Molecular mechanisms of RNA-induced gene silencing
-siRNA and microRNAs -Short, double stranded ribonucleotides -siRNAs (short interfering RNAs): Arise in cell due to virus infection-produce double stranded RNA which is recognized and cleaved by dicer -microRNAs: noncoding RNAs transcribed from the genome that negativley regulate gene expression. -siRNAs are derived from longer RNA molecules that are linear, double stranded, and located in the cell cytoplasm. In nature, these siRNA precursors arise within cells as a result of virus infection or the expression of transposons- both of which synthesize double stranded RNA molecules as part of their life cycles. In the cytoplasm, double stranded RNA molecules are recognized by an enzyme complex known as dicer which are cleaved by diver into siRNAs. -miRNAs are derived from single stranded RNAs that are transcribed within the nucleus from the cell's own genome and contain a double stranded stem loop structure. Nuclease enzymes within the nucleus recognize these step loop structures and cleave them from the longer single stranded RNA. These fragments then are exported to the cytoplasm where they are further processed by the dicer complex into short, linear double stranded miRNAs.
Three pathways of mRNA degration
1.) Enzymes shorten length of poly A tail ex: poly(A) ribonuclease (PARN) Binding of poly A binding protein to tails stabilizes mRNA 2.) decapping enzymes removes 7-methyl guanine cap- mRNA now unstable 3.) Endonuclease cleaves mRNA internally -Enzymes may be targeted for degradation by enzymes that shorten the length of the poly a tail. In newly synthesized mRNAs, the poly a tail is about 200 nucleotides long and binds the protein poly a binding protein. the binding of this protein helps stabilize the mRNA. if the poly a tail is shorten to less than 30 nucleotides, the mRNA becomes unstable and acts as a substrate for exonucleases that degrade the RNA in either 5'-3' or 3'-5' direction. -mRNA may be cleaved internally by an endonuclease, providing unprotected ends at which exonuclease degradation will proceed.
