REPAIRhttp://www.unc.edu/courses/2009spring/envr/740/001 slide 13Begin 02/12/09Factors that are required for initiation along with the RNA polymerase comprise the “basal transcription apparatus”. There are also factors in addition to the basal apparatus that modulate efficiency and are characteristic for specific promoters. There are also inducible factors that function as upstream factors, but, as the term inducible implies, are present only at specific timesor synthesized only under certain circumstances and therefore have a regulatory role. One additional distinction between eukaryotes and prokaryotes is the presence of units called enhancers associated with eukaryotic transcription. Enhancers are sequences that, like promoters contain binding sites for factors that function to facilitate transcription. But they are by definition located distant from the promoter, may be either upstream or downstream from the start. Typically, recognition/binding sites have a much higher density in enhancers than in promoters. Where there are relatively few sites that are affected by mutations in promoters, transcription is down-regulated by mutation of a large number of sites within an enhancer. This isillustrated in the next overhead, which shows a histogram representation of a saturation mutation study on an enhancer:[OH; enhancer]Binding sites in the enhancer may also be elements in promoters, and we have already mentionedthe octamer which is appearing here in an inverted orientation. The complex assembled at the enhancer does not interact directly with the polymerase, but seems to aid in assembling the pre-initiation complex at the promoter. Since the proteins of the initiation complex are physically associated, the location of elements at large distances from the promoter sequence implies that the complexed DNA must be bent into loops in order to allow factors to associate.One additional strategy of eukaryotic cells with regard to transcriptional regulation is methylation. In eukaryotes, the state of methylation of C in CpG doublets near the 5 end of some genes appears to be correlated with gene expression. Genes are transcribed in the absence of methylated C in 5′-CpG-3′ doublets. In addition, some transcribed genes have unmethylated 1CpG-rich “islands” in the promoter region extending into the beginning of transcribed DNA. Theislands are areas with a particularly dense occurrence of CpG doublets, having a G•C content up to 60% compared to 40% in bulk DNA. The islands are associated with genes that are constitutively transcribed, supporting a role in gene expression. The term used to describe undermethylation is “hypomethylation”. At this point, the mechanism for the observed correlation of expression with hypomethylation is not known, but one observation is that in the absence of methylated CpG doublets, DNA is less condensed and accessible to transcription factors and RNA polymerases. Hypomethylation does not in all cases lead to expression, so at the moment hypomethylation is considered to give a gene the potential for being expressed, but does not guarantee expression. We mentioned briefly that methylation status may be affected by diet or other environmentally imposed conditions and the consequences of changes in gene expression induced in this manner comprise “epigenetic” phenomena.In contrast to the prokaryotic the lac operon, where all genes relevant to utilization of β-galactosides were clustered together and transcribed serially under the control of a single promoter, eukaryotic genes are generally not clustered in this way, so there must be a mechanismto coordinate transcription of proteins having related functions. The need for such a mechanism is reflected in differences in how gene expression is regulated. Another important point is that our description of the initiation of eukaryotic transcription represented a basal, constitutive level.However, as we just implied, many eukaryotic genes are inducible, implying the requirement for control elements in addition to the basal elements. A mechanism to accomplish both the coordination of transcription of genes under control of separate promoters and to provide for inducability lies in sequences called response elements, which are short domains recognized by specific inducible transcription factors. Such response elements may be located in promoters or enhancers. The following overhead is a schematic of the regulatory region of the human metallothionein gene.[OH; regulatory region of metallothionein gene]Acronyms in the overhead are:BLE=basal level element2TRE=phorbol ester response elementMRE= metal response element – activated by presence of heavy metalsGRE= glucocorticoid response elementPoint out that the GRE and TRE elements may indicate multiple inducibilities or a role for steroids in mediating the metallothionein response.Having now had additional background on eukaryotic transcription, we will return to the initiation process to describe some aspects that will be useful later. The next slide shows DNA binding motifs commonly associated with transcription factors, which we briefly alluded to in describing the lac repressor and the TBP.Of the transcription factors, a sub-class of Zn finger-containing factors is activated by complexation with steroids and has received a great deal of attention. Other specific DNA binding motifs associated with other families of transcription factors have been identified are shown below:[OH; Table of receptors and zinc finger motif]Zn fingersHelix-turn-helix (we already encountered this motif in DNA binding domains of the lac repressor).Helix-loop-helixThe Leu zipper motif is included in this overhead because it often present near the DNA binding domain, and serves as mechanism for association of transcription factors to form dimers, a necessary interaction required for activation of many transcription factors.The generic zinc finger motif is shown at the bottom of the slide.-Cys-X2-4-Cys-X3-Phe-X5-Leu-X2-His-X3-His-3The convention in describing motifs is that the amino acids that are indicated by name are highlyconserved, other residues are indicated by a generic “X”. For Zn fingers, the Cys, His residues are the structural organization components- the SH (sulfhydryl) groups of Cys and the N of His efficiently bind Zn2+. The next slide shows the organization of the protein and a cartoon of DNA binding.[OH; Zn finger binding-incl. motif in fig.]Crystal