Year III Pharm.D Dr. V. Chitra 1
Genome entire genetic material of an individual Transcriptome set of transcribed sequences Proteome set of proteins encoded by the genome 2
Only one strand of DNA serves as a template for transcription. Different genes are transcribed from different strands 3
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5 - Promoter Exon1 UTR Intron1 Exon2 Terminator 3 splice UTR splice transcription Poly A translation protein 5
Promoter CDS UTR Terminator UTR Genomic DNA transcription mrna translation protein 6
Promoter determines: 1. Which strand will serve as a template. 2. Transcription starting point. 3. Strength of polymerase binding. 4. Frequency of polymerase binding. 7
One type of RNA polymerase. Pribnow box located at 10 (6-7bp) 35 sequence located at -35 (6bp) 8
3 types of RNA polymerases are employed in transcription of genes: RNA polymerase I transcribes rrna RNA polymerase II transcribes all genes coding for polypeptides RNA polymerase III transcribes small cytoplasmatic RNA, such as trna. 9
Goldberg-Hogness or TATA located at 30 Additional regions at 100 and at 200 Possible distant regions acting as enhancers or silencers (even more than 50 kb). 10
Promoters sequences can vary tremendously. RNA polymerase recognizes hundreds of different promoters 11
Strong promoter resemble the consensus sequence. Mutations at promoter sites can influence transcription. Human gene Beta globin 12
Conclusions: 1. Promoters are very hard to predict. 2. Promoter prediction must be organism- dependent (and even polymerasedependent). 13
The newly synthesized mrna forms a stem and loop structure (lollipop). A disassociation signal at the end of the gene that stops elongating and releases RNA polymerase. All terminators (eukaryotes and prokaryotes) form a secondary structure. 14
The terminator region pauses the polymerase and causes disassociation. 15
Eukaryotics only Removing internal parts of the newly transcribed RNA. Takes place in the cell nucleus (hnrna) 16
Conserved splice sites are shared by both the exon and the intron. Different signals on the donor site (3 ) and on the acceptor site (5 ). 17
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Different splice patterns from the same hnrna sequence. Different products from the same gene Different organs, different stages of development in the same cell. Exact splice sites are difficult to predict 19
GENE EXPRESSION
Every cell has the same DNA and therefore the same genes. But different genes need to be on and off in different types of cells. Therefore, gene expression must be regulated. liver embryo muscle bone sperm (The first statement on this slide is not completely true. Which of these cells does not have exactly the same DNA as the other? Can you think of any other examples of cells in your body that have different DNA than most of the others?)
Gene expression must be regulated in several different dimensions In time: 10 wks 6 mos 14 wks 1 day 12 mos 18 mos At different stages of the life cycle, different genes need to be on and off. M. Halfon, 2007
Importance of gene regulation common variation behavior pattern evolution chromosome inactivation metabolism pathology (mutation) M. Halfon, 2006
Gene Regulation and Nutrition: Development (organs, cell types) muscle liver (diseased) fat embryo embryo brain intestines With respect to nutrition, gene regulation is important to guide the development of organs, tissues, and cell types required to ingest, digest, and metabolize nutrients.
Genes can be regulated at many levels DNA TRANSCRIPTION RNA TRANSLATION PROTEIN The Central Dogma
Control of Gene Expression Transcription Factors Transcription factors (TFs) are proteins that bind to the DNA and help to control gene expression. We call the sequences to which they bind transcription factor binding sites (TFBSs), which are a type of cis-regulatory sequence.
Determining Transcription Factor Binding Sites DNAseI footprinting, which takes advantage of the ability of the enzyme DNAseI to non-specifically cleave DNA. A bound TF protects the DNA from cleavage, leaving a visible footprint when the digested DNA is visualized by gel electrophoresis. Figure 8-54. The DNA footprinting technique. (A) This technique requires a DNA molecule that has been labeled at one end (see Figure 8-24B). The protein shown binds tightly to a specific DNA sequence that is seven nucleotides long, thereby protecting these seven nucleotides from the cleaving agent. If the same reaction were performed without the DNA-binding protein, a complete ladder of bands would be seen on the gel (not shown). (B) An actual footprint used to determine the binding site for a human protein that stimulates the transcription of specific eucaryotic genes. These results locate the binding site about 60 nucleotides upstream from the start site for RNA synthesis. The cleaving agent was a small, iron-containing organic molecule that normally cuts at every phosphodiester bond with nearly equal frequency. (B, courtesy of Michele Sawadogo and Robert Roeder.) Source: Alberts et al., Molecular Biology of the Cell
Determining Transcription Factor Binding Sites Other methods include - EMSA (gel shift) - SELEX (Systematic Evolution of Ligands by EXponential enrichment) -protein-binding microarrays -ChIP-chip/ChIP-seq
Control of Gene Expression Transcription Factors Most transcription factors can bind to a range of similar sequences. We call this a binding motif. Wasserman, W. W. and A. Sandelin (2004). Nat Rev Genet 5(4): 276-287.
Control of Gene Expression Transcription factor binding sites are found within larger functional units of the DNA called cis-regulatory elements. There are two main type of cis-regulatory elements: promoters, and cis-regulatory modules (sometimes called enhancers ). cis-regulatory module (CRM) TFBS transcription factor binding site (TFBS) TFBS Image adapted from Wolpert, Principles of Development
Control of Gene Expression: Promoters Every gene has a promoter, the DNA sequence immediately surrounding the transcription start site. The promoter is the site where RNA polymerase and the so-called general transcription factors bind.
Control of Gene Expression: CRMs Additional gene regulation takes place via the cis-regulatory modules (CRMs), which can be located 5 to, 3 to, or within introns of a gene. CRMs can be very far away from the gene they regulate over 50 kb and other genes might even lie in between! cis-regulatory module (CRM) TFBS TFBS transcription factor binding site (TFBS)
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Protein Synthesis (Gene Expression) Notes Proteins (Review) Proteins make up all living materials
Proteins are composed of amino acids there are 20 different amino acids Different proteins are made by combining these 20 amino acids in different combinations
Proteins are manufactured (made) by the ribosomes
Function of proteins: 1. Help fight disease 2. Build new body tissue 3. Enzymes used for digestion and other chemical reactions are proteins (Enzymes speed up the rate of a reaction) 4. Component of all cell membranes
Making a Protein Transcription First Step: Copying of genetic information from DNA to RNA called Transcription Why? DNA has the genetic code for the protein that needs to be made, but proteins are made by the ribosomes ribosomes are outside the nucleus in the cytoplasm. DNA is too large to leave the nucleus (double stranded), but RNA can leave the nucleus (single stranded).
Part of DNA temporarily unzips and is used as a template to assemble complementary nucleotides into messenger RNA (mrna).
mrna then goes through the pores of the nucleus with the DNA code and attaches to the ribosome.
Making a Protein Translation Second Step: Decoding of mrna into a protein is called Translation. Transfer RNA (trna) carries amino acids from the cytoplasm to the ribosome.
These amino acids come from the food we eat. Proteins we eat are broken down into individual amino acids and then simply rearranged into new proteins according to the needs and directions of our DNA.
A series of three adjacent bases in an mrna molecule codes for a specific amino acid called a codon. A triplet of nucleotides in trna that is complementary to the codon in mrna called an anticodon. Each trna codes for a different amino acid. Amino acid Anticodon
mrna carrying the DNA instructions and trna carrying amino acids meet in the ribosomes.
Amino acids are joined together to make a protein. Polypeptide = Protein
Protein Synthesis