Chap. 7 Transcriptional Control of Gene Expression (Part A)

1. Chap. 7 Transcriptional Control of Gene Expression (Part A) • Topics • Control of Gene Expression in Bacteria • Overview of Eukaryotic Gene Control and RNA Polymerases • Goals • Introduce the wide variety of mechanisms used for gene control. • Learn the properties of RNA polymerases. Transcriptionally active polytene chromosomes

2. Mutations in Transcription Factors The protein factors that regulate gene expression via transcription are critical to many biological processes. When these factors function incorrectly, pathological and other aberrant effects occur (Fig. 7.1). Examples shown include development of the eye in humans, wing development in flies, and flower development in plants.

3. Overview of Eukaryotic Transcription Control The regulation of gene expression, or "gene control" is concerned with all possible ways in which gene activity can be controlled. While regulation of transcription initiation is the most common method of control, alternative splicing, etc. are commonly used as well. As shown in Fig. 7.2, control of transcription initiation can be achieved by activators and repressors that modulate chromatin condensation state. Many other protein factors (covered below) must function correctly so that transcription occurs at the appropriate rate for proper biological function.

4. The E. colilac Operon The control of gene expression by transcription activation and repression has been studied extensively in bacteria. As an example, the E. coli lac operon, which encodes 3 genes (lacZYA) involved in lactose metabolism, uses both mechanisms of control (Fig. 7.3). A specific repressor protein (the lac repressor) inhibits transcription from the lac promoter by binding to an adjacent DNA sequence known as the lac operator in the absence of lactose. A general activator protein known as catabolite activator protein (CAP) binds to a site immediately upstream of the promoter, stimulating transcription. However, the binding of CAP to its site requires the co-activator, cAMP, the concentration of which is low in glucose-containing medium. The cellular conditions under which these two regulatory proteins bind to the lac promoter region are illustrated in the next slide.

5. Regulation of lac Operon Transcription In the presence of glucose and absence of lactose, the transcription of the lac operon is repressed (Fig. 7.3a). The lac repressor is bound to the lac operator and CAP is not bound to its control site due to low levels of cAMP. The addition of the inducer lactose to media and its binding to the lac repressor causes the repressor to undergo a conformational change and dissociate from the lac operator sequence (Fig. 7.3b). However, if glucose still is present in the medium, CAP cannot bind to its site and transcription is relatively low. Finally, in the absence of glucose and presence of lactose, cAMP levels become high. cAMP binds to CAP, and the complex binds to the control site, strongly activating transcription by RNA polymerase (Fig. 7.3c). E. coli contains seven sigma factors (e.g., s70,s54) that are needed by RNA polymerase for recognition of promoters and transcription initiation. The lac promoter requires s70, which is the most commonly used  factor in this bacterium.

6. Enhancer Control of Bacterial Transcription s70 combines with RNA polymerase prior to DNA binding. The alternative sigma factor, s54, acts very differently in that it binds to its promoters in the absence of the polymerase. s54-RNA polymerase further requires activators that bind to upstream enhancer sequences for initiation. As shown in Fig. 7.4, these "enhancer binding proteins" (e.g., NtrC) contact s54-RNA polymerase by looping of the DNA between them. The mechanism of transcription activation by 54-RNA polymerase is similar to transcription activation in eukaryotes.

7. Regulatory Elements in Eukaryotic Genes The regulation of transcription of many eukaryotic genes is highly complex. Genes can be expressed differently in various tissues, during different stages of development, and under different environmental conditions. The complexity of expression of the Pax6 gene, which is important for development of the eye and other tissues, is illustrated in Fig. 7.7. Different versions of the Pax6 protein are expressed in different tissues and times of development in mouse embryos (right). Differential gene expression is achieved via DNA regulatory sequences located upstream, within, and even downstream of genes (left). These regulatory sequences (promoters, enhancers, etc.) are bound by varying sets of transcription factors in different tissues.

8. Eukaryotic RNA Polymerases Bacteria contain only one RNA polymerase. This enzyme has a aIII ß ß’ w subunit composition (Fig. 7.11). Eukaryotes contain three nuclear RNA polymerases that are larger, but have core subunits homologous to those in the bacterial enzyme. The RPB1 subunit of RNA Pol II has a C-terminal domain (CTD) that is important for regulation of transcription. The CTD contains a 7-amino-acid unit that is repeated 52 times. This sequence is unphosphorylated prior to initiation, but is phosphorylated on serine and tyrosine residues after transcription initiates. Pol I transcribes pre-rRNA, which is processed into the 28S, 18S, and 5.8S species. Pol II transcribes mRNA, miRNAs, and 5 snRNAs. Pol III transcribes tRNA, 5S rRNA, and 1 snRNA.