Lecture 9 Controlling gene expression BIOLOGY Campbell, Reece and Mitchell Chapter 18 334- (352-356) Every cell in your body contains the same number of genes approximately 35, 000 DNA is wound around histone proteins chromatin eukaryotes But in any given tissue or cell type most of these genes are turned off i.e. they are not being expressed In different cell types, different cell-specific sets of genes are turned on i.e. are expressed e.g. in mature red blood cells very few genes are expressed A single gene coding for hemoglobin is expressed at very high levels in these cells. 1
www.chromatintoronto.ca/projects.html Gene expression is highly regulated Many aspects of gene expression are very tightly regulated e.g. When and for how long a gene will be expressed e.g. some genes are expressed for only a few hours during development of the embryo and are never expressed in the adult Where a gene will be expressed i.e. in what cell and tissue types e.g. hemoglobin is only expressed in one cell type the red blood cells How much expression i.e. low or high levels of expression In plants the RUBISCO gene plays a major role in photosynthesis It codes for the most abundantly expressed protein in leaves = 50% of the total protein i.e. the gene is very highly expressed 2
The expression of many genes may also be regulated in response to external factors e.g. the gene coding for RUBISCO shows two types of regulation: (1) Regulation by light RUBISCO is only expressed if leaves are exposed to light there is no expression in the dark (2) Tissue-specific regulation RUBISCO is only expressed in green tissues (e.g. leaves) - it is not expressed in root tissue (even if exposed to light) Hox Genes Control development Hox genes need to be highly regulated to get expressed at the right time and correct level to orchestrate mammalian development in utero. Mario Capecchi won the Nobel Prize in 2007 for his research on Hox genes & their role in defining the mammalian development plan. Individual Hox genes were mutated in mice and the effects of the mutations observed. In TCD last week at History Society! 3
How might gene expression be regulated? There are several potential control points in the gene expression pathway: 1. Control of transcription 2. Control via mrna processing (not in bacteria) 3. Control of translation www.google.ie/imgres?imgurl=http://course1.winona.edu/sberg/illust/introns.jpg 4
ww.google.ie/imgres?imgurl=http://gcat.davidson.edu/mediawiki-1.15.0/images/e/e1/eukaryotic.jpg&imgrefurl=http://gcat.davidson.edu/mediawiki-1.15.0/index.php/post- transcriptional_regulation_technologies_- _Erin_Zwack&usg= CNiMsnSqWcsTc79Ltb2qTTYq1iA=&h=683&w=600&sz=62&hl=en&start=24&zoom=1&tbnid=6aBZYKt9b8l1dM:&tbnh=139&tbnw=122&ei=3XJvUozDJO_n7AaO0oDo CQ&prev=/search%3Fq%3Dcontrol%2Bof%2Bgene%2Bexpression%2Bin%2Beukaryotes%26start%3D20%26um%3D1%26sa%3DN%26hl%3Den%26gbv%3D2%26tbm %3Disch&um=1&itbs=1&sa=X&ved=0CDAQrQMwAzgU 3 bases of DNA (a codon) codes for 3 bases of RNA which codes for a single amino acid. The last codon of a run of codons coding for amino acids is called a stop codon a signal to stop coding. Mature RNAs (mrnas) without a stop codon are degraded rapidly the cell has methods to get rid of aberrant RNAs the cell is always double checking things! Dr. Ambro van Hoof, University of Texas,, Houston 5
sandwalk.blogspot.com prokayotic gene.jpg faculty.ksu.edu.san noncoding.gens.jpg While approx. 95% of DNA does not code for protein, we are now realising that approx. 1/3 of DNA in the mammalian genome codes for non-coding RNAs (that is they never get translated into proteins). It turns out that these non-coding RNAs are important in regulating gene expression. One class of non-coding RNAs involved in this is micrornas. 6
Transcription in bacteria - prokaryotes 1. Transcription requires the enzyme RNA polymerase 2. To initiate transcription, RNA polymerase must bind to a special DNA sequence at the start of the gene called a promoter. RNA pol promoter gene 3. If RNA polymerase is prevented from binding to the promoter, transcription will not occur Inducible gene expression in bacteria Bacteria can adapt their metabolism to take advantage of nutrients in the environment E. coli prefers glucose to all other sugars as a carbon source However, if no glucose is available, it can use other sugars like the sugar present in milk = lactose E. coli can respond very rapidly, within 15 minutes, to take advantage of the availability of lactose It responds by rapidly expressing 3 genes which code for enzymes that are required for lactose metabolism Expression of these genes is said to be induced by lactose 7
The 3 enzymes required for lactose metabolism are translated from the single mrna transcript: The enzymes are: (i) Lactose permease which is encoded by the lacy gene It functions as a membrane transporter which imports lactose from outside the cell (ii) Beta-galactosidase which is encoded by the lacz gene It functions to hydrolyse lactose to the monosaccharides glucose and galactose (iii) Transacetylase which is encoded by the laca gene It precise function is not fully known, howevr it an enzyme that transfers an acetyl group from acetyl-coa to β-galactosides The 3 genes: lacz, lacy and laca are found side-by-side as a single transcription unit This means that a single mrna molecule is transcribed DNA mrna lacz lacy laca lacz lacy laca This type of gene organization, where genes that function in the same metabolic pathway are grouped together is called an operon Because these genes are all involved in lactose metabolism this group is called the lactose operon or the lac operon 8
Lactose induces expression of the lac operon A. If lactose is not present in the environment: The lac operon is not being actively transcribed (i.e. turned OFF) The 3 enzymes required for lactose metabolism are present in extremely low amounts B. If lactose is present in the environment: Transcription of the lac operon is induced Within 15 minutes expression of the operon increases by approximately 1000 fold How is the lac operon turned OFF if lactose is absent? How does lactose induce expression of the lac operon? Answers: 1. The operon contains a 4 th gene called laci that codes for a regulatory protein (not an enzyme!) 2. This protein functions as a repressor of transcription 3. The repressor binds to a DNA sequence called the operator located between the promoter and the 3 genes lacz, lacy, laca 4. When the repressor is bound to the operator, RNA polymerase cannot bind to the promoter. 9
The repressor stays bound to the operator sequence as long as lactose is absent. This prevents RNA polymerase from binding to the promoter LacI lacy laca LacI Induction of transcription by lactose Lactose binds to repressor and changes its shape. Repressor can no longer bind to the operator. This allows RNA polymerase to bind to the promoter and begin transcription promoter operator 10
Summary In bacteria, genes that carry out related functions are usually grouped together to form a regulatory unit the operon Operons contain at least one gene whose protein product functions to regulate transcription of the operon In the case of the lac operon, the laci gene codes for a repressor protein that is normally found bound to the operator Transcription of the lac operon is inducible by the sugar lactose If lactose is present, it binds to the repressor and changes its affinity for the operator. When the repressor vacates the operator, RNA polymerase can bind to the promoter and begin transcription. 11
(EM: David E and Ada L Olins) Eukaryotes Mimimal Transcriptional Machinery Minimal complex FJ Asturias 2004 Current Opinion in Structural Biology 14: 121-129 RNA polymerase II, Transcription factor IIF Transcription factor IIB TATA binding protein TBP Minimal complex capable of promoter directed initiation 12
Nature Reviews Vaquerizas et al. April 2009 Nature Reviews Vaquerizas et al. April 2009 Hox gene clusters on chromsomes 2, 7, 12 & 17 13