Part 5. Eukaryotic gene expression Chapter 25
Regulating Eukaryotic Transcription
25.1 Introduction
expression of Eukaryotic genes might be regulated at any one of several stages:We can distinguish (at least) five potential control points, forming the series: Activation of gene structure Initiation of transcription Processing the transcript Transport to cytoplasm Translation of mRNA激活、起始转录、加工、运 输、翻译 Figure 22.1 Gene expression is controlled principally at the initiation of transcription, and it is rare for the subsequent stages of gene expression to be used to determine whether a gene is expressed, although control of processing may be used to determine which form of a gene is represented in mRNA.
Positive clones confirmed on SD/-Ade/-HisHale Waihona Puke -Leu/-Trp/X-a-Gal
25.5 Response elements are recognized by activators
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A response element is a sequence in a eukaryotic promoter or enhancer that is recognized by a specific transcription factor. The heat shock response element (HSE) is a sequence in a promoter or enhancer that is used to activate a gene by an activator induced by heat shock. The glucocorticoid response element (GRE) is a sequence in a promoter or enhancer that is recognized by the glucocorticoid receptor ,which is activated by glucocorticoid steroids. The serum response element (SRE) is a sequence in a promoter or enhancer that is activated by transcription factor(s) induced by treatment with serum. This activates genes that stimulate cell growth.
25.3 Independent domains bind DNA and activate transcription
Activators and other regulatory proteins require two types of ability: •They recognize specific target sequences located in enhancers, promoters, or other regulatory elements that affect a particular target gene. •Having bound to DNA, an activator exercises its function by binding to other components of the transcription apparatus.
25.3 Independent domains bind DNA and activate transcription
key concepts:
•DNA-binding activity and transcription-activation are carried by independent domains of an activator. •The DNA-binding domain determines specificity for the target promoter or enhancer. •The role of the DNA-binding domain is to bring the transcriptionactivation domain into the vicinity of the promoter.
25.2 There are several types of transcription factors
Initiation of transcription involves many protein-protein interactions among transcription factors bound at the promoter or at an enhancer as well as with RNA polymerase. We can divide the factors required for transcription into several classe: Basal factors, together with RNA polymerase, bind at the startpoint and TATA box. Activators are transcription factors that recognize specific short consensus elements. They bind to sites in the promoter or in enhancers. Another group of factors necessary for efficient transcription do not themselves bind DNA. Coactivators provide a connection between activators and the basal apparatus. Some regulators act to make changes in chromatin.
25.3 Independent domains bind DNA and activate transcription
Figure 25.3 DNA-binding and activating functions in a transcription factor may comprise independent domains of the protein.
25.3 Independent domains bind DNA and activate transcription
The activator GAL4 has a DNA-binding domain that recognizes a UAS, and an activating domain that stimulates initiation at the target promoter. The bacterial repressor LexA has an N-terminal DNAbinding domain that recognizes a specific operator. When LexA binds to this operator, it represses the adjacent promoter. Figure 22.4 The ability of GAL4 to activate transcription is independent of its specificity for binding DNA. When the GAL4 DNA-binding domain is replaced by the LexA DNA-binding domain, the hybrid protein can activate transcription when a LexA operator is placed near a promoter.
Construct pathogen induced yeast twohybrid cDNA library
cDNA library of oilseed rape line RS-1 constructed in yeast
Library Insert size analysis by PCR
Yeast cells that are mating. A zygote typically has three-lobed shape, the lobes representing the two haploid(parental) cells and the budding diploid cells
25.2 There are several types of transcription factors
Figure 22.2 Factors involved in gene expression include RNA polymerase and the basal apparatus, activators that bind directly to DNA at the promoter or at enhancers, co-activators that bind to both activators and the basal apparatus, and regulators that act on chromatin structure.