1. Liver Cells Cartilage Cells Red Blood Cells Stem Cells What makes cells from the same individual look different? DNA sequence in each cell is the same, but different cell types have different “GENE EXPRESSION PATTERNS”

2. When a gene is “on” and its protein or RNA product is being made, scientists say that the gene is being EXPRESSED. The on and off states of all of a cell’s genes is known as a GENE EXPRESSION PROFILE. Each cell type has a unique gene expression profile. InsulinDNA?Protein? Muscle Cell X Pancreatic Cell

3. KEY CONCEPT Gene expression is carefully regulated in both prokaryotic and eukaryotic cells. http://www.cpalms.org/Public/PreviewResourcePV/Preview/145675 Gene Expression and Regulation

4. Gene Regulation in Prokaryotes Bacteria adapt to changes in their surroundings by using regulatory proteins to turn groups of genes on and off in response to various environmental signals. The DNA of Escherichia coli is sufficient to encode about 4000 proteins, but only a fraction of these are made at any one time. E. coli regulates the expression of many of its genes according to the food sources that are available to it.

5. Prokaryotic cells turn genes on and off by controlling transcription. An operon is a cluster of bacterial genes along with an adjacent promoter that controls the transcription of those genes. An operon includes a promoter, an operator, and one or more structural genes that code for all the proteins needed to do a job. A promotor is a DNA segment that allows a gene to be transcribed. An operator is a part of DNA that turns a gene “on” or ”off.”

6. The Lac Operon –Operons are most common in prokaryotes. –The lac operon was one of the first examples of gene regulation to be discovered. –The lac operon has three genes that code for enzymes that break down lactose.

7. The lac operon acts like a switch. –The lac operon is “off” when lactose is not present. –The lac operon is “on” when lactose is present.

8. When the genes in an operon are transcribed, a single mRNA is produced for all the genes in that operon. This mRNA is said to be polycistronic because it carries the information for more than one type of protein. The operator is a short region of DNA that lies partially within the promoter and that interacts with a regulatory protein that controls the transcription of the operon.

9. Here's an analogy. A promoter is like a doorknob, in that the promoters of many operons are similar. An operator is like the keyhole in a doorknob, in that each door is locked by only a specific key, which in this analogy is a specific regulatory protein.

10. The regulatory gene lacI produces an mRNA that produces a Lac repressor protein, which can bind to the operator of the lac operon. Regulatory genes are not necessarily close to the operons they affect. The general term for the product of a regulatory gene is a regulatory protein. The Lac regulatory protein is called a repressor because it keeps RNA polymerase from transcribing the structural genes. Thus the Lac repressor stops transcription of the lac operon

11. In the absence of lactose, the Lac repressor binds to the operator and keeps RNA polymerase from transcribing the lac genes. –It would be energetically wasteful for E. coli if the lac genes were expressed when lactose was not present.

12. The effect of the Lac repressor on the lac genes is referred to as negative regulation. –When lactose is present, the lac genes are expressed because allolactose binds to the Lac repressor protein and keeps it from binding to the lac operator. –Allolactose is an isomer of lactose. Small amounts of allolactose are formed when lactose enters E. coli. –Allolactose binds to an site on the repressor protein causing it to change shape. As a result of this change, the repressor can no longer bind to the operator region and falls off. RNA polymerase can then bind to the promoter and transcribe the lac genes.

13. Allolactose is called an inducer because it turns on, or induces the expression of, the lac genes. The presence of lactose (and thus allolactose) determines whether or not the Lac repressor is bound to the operator.

14. –Allolactose binds to an site on the repressor protein causing it to change shape. –As a result of this change, the repressor can no longer bind to the operator region and falls off.

15. –RNA polymerase can then bind to the promoter and transcribe the lac genes. –When the enzymes encoded by the lac operon are produced, they break down lactose and allolactose, eventually releasing the repressor to stop additional synthesis of lac mRNA.

16. Messenger RNA breaks down after a relatively short amount of time. Whenever glucose is present, E. coli metabolizes it before using alternative energy source such as lactose. Glucose is the preferred and most frequently available energy source for E. coli. The enzymes to metabolize glucose are made constantly by E. coli. When both glucose and lactose are available, the genes for lactose metabolism are transcribed at low levels. Only when the supply of glucose has been exhausted does does RNA polymerase start to transcribe the lac genes efficiently, which allows E. coli to metabolize lactose. When both glucose and lactose are present, the genes for lactose metabolism are transcribed to a small extent

17. Eukaryotes regulate gene expression at many points. Different sets of genes are expressed in different types of cells. Transcription is controlled by regulatory DNA sequences and protein transcription factors.

18. –Most eukaryotes have a TATA box promoter. –This is the sequence RNA polymerase recognizes and binds to: how it “knows” where a gene starts

19. –Enhancers and silencers speed up or slow down the rate of transcription. –Each gene has a unique combination of regulatory sequences.

20. RNA processing is also an important part of gene regulation in eukaryotes. mRNA processing includes three major steps. Eukaryotic mRNA Processing

21. mRNA processing includes three major steps. –Introns are removed and exons are spliced together. –A methyl cap is added. –A poly-A tail is added.

22. Unlike prokaryotes which have one RNA polymerase that makes all classes of RNA molecules, eukaryotic cells have three types of RNA polymerase (called RNA pol I, RNA pol II, and RNA pol III), and each type of RNA is made by its own polymerase: –RNA polymerase I makes ribosomal RNA (rRNA) –RNA polymerase II makes messenger RNA (mRNA) –RNA polymerase III makes transfer RNA (tRNA)

23. RNAs are made in the nucleus of a eukaryotic cell, but function in protein synthesis in the cytoplasm. Unlike prokaryotic mRNAs, eukaryotic mRNAs undergo extensive modifications after synthesis by RNA polymerase II. These changes include capping, polyadenylation, and splicing.

24. –Modification of the 5'-ends of eukaryotic mRNAs is called capping. –The cap consists of a methylated GTP. –Capping occurs very early during the synthesis of eukaryotic mRNAs, even before mRNA molecules are finished being made by RNA polymerase II. –Capped mRNAs are very efficiently translated by ribosomes to make proteins. –In fact, some viruses, such as poliovirus, prevent capped cellular mRNAs from being translated into proteins. This enables poliovirus to take over the protein synthesizing machinery in the infected cell to make new viruses. Adding the Methyl Cap

25. Modification of the 3'-ends of eukaryotic mRNAs is called polyadenylation. Polyadenylation is the addition of several hundred A nucleotides to the 3' ends of mRNAs. This string of A’s makes the poly-A tail. Polyadenylation (Poly-A tail)

26. –Eukaryotic genes are often interrupted by sequences that do not appear in the final RNA. –The intervening sequences that are removed are called introns. –The process by which introns are removed is referred to as splicing. –The sequences remaining after the splicing are called exons. –Although most higher eukaryotic genes have introns, some do not. Splicing

27. –Higher eukaryotes tend to have a larger percentage of their genes containing introns than lower eukaryotes, and the introns tend to be larger as well. –The pattern of intron size and usage roughly follows the evolutionary tree, but this is only a general tendency. –The human titin gene has the largest number of exons (178), the longest single exon (17,106 nucleotides) and the longest coding sequence (80,781 nucleotides = 26,927 amino acids). –The longest primary transcript, however, is produced by the dystrophin gene (2.4 million nucleotides).

28. Splicing has evolutionary implications. –Exons often coincide with protein "domains”. –Domains are parts of the protein with a specific function. –Exons can be readily "exchanged" between different genes by recombination. –This means that new types of proteins can be formed relatively easily. Usefulness of splicing

29. Splicing also allows a cell to "swap" exons during gene expression. –For example, during development, some genes are spliced one way, and then spliced a different way later. –Changing the way a mRNA is spliced changes the amino acid sequence in the protein made from it, so cells can in this way "modify" the sequence, and function, of a protein. –Splicing is yet another mechanism for regulating whether or not a specific version of a protein is made, how much of it is made, and when it is made.

30. One very good example of exon shuffling can be seen in the tropomyosin gene. Tropomyosin is a protein involved in muscle-like contraction in cells. It is present in many different types of cells of the body. The tropomyosin mRNAs in different types of muscle cells are slightly different from each other, but all come from the same gene.

31. During the research of Andrew Fire and Craig Mello on gene expression in the worm C. elegans, they found that injecting mRNA that encodes for muscle protein production elicited no responses from the worms. –Bear in mind that the genetic code in the mRNA is considered as the sense sequence. Discovery of RNA Interference or RNAi

32. –They also tried to inject antisense RNA into the worms which can pair with the sense sequence mRNA but it also elicited no responses from the worms. –Finally, when they tried to inject both the sense and the antisense RNA together, they noted twitching movements from the worms. These results surprised them since they know that the same kinds of movements were noted from worms whose genes encoding for muscle protein were dysfunctional.

33. To explain the results that they got, Fire and Mello hypothesized that the double-stranded RNA molecule formed by the binding of the sense and antisense RNA silences the gene carrying exactly the same code as the RNA molecule. To test their hypothesis, they injected double- stranded RNA that codes for specific proteins. In all their experiments, they found that the genes carrying exactly the same code as the RNA they injected were silenced.

34. Their discovery on RNA interference is noteworthy for two reasons.RNA interference –First, with RNAi, researchers can specifically knockdown the production of any protein in a cell. –Second, initially scientists thought that a portion of the DNA called introns were just junk DNA and they serve very little purpose, buy now they know that much of these introns code for RNAi elements.

35. Small interfering RNA (siRNA) are small pieces of double-stranded (ds) RNA that can be used to "interfere" with the translation of proteins by binding to and promoting the breakdown of messenger RNA (mRNA) at specific sequences. In doing so, they prevent the production of specific proteins.translationRNA The process is called RNA interference (RNAi), and may also be referred to as siRNA silencing or siRNA knockdown.RNA interference siRNA

36. –siRNA often comes from vectors, like viruses, and have been found to play a role in antiviral defense, degradation of over-produced mRNA or mRNA for which translation has been aborted, and preventing disruption of genomic DNA by transposons.vectorsDNA –The siRNA then "seeks out" an appropriate target mRNA, where the siRNA then causes the mRNA to be broken down. Many diseases can potentially be treated by inhibiting gene expression. –Therefore, the design of synthetic siRNA for therapeutic uses has become a popular objective of many biopharmaceutical companies. therapeutic