1. 1 Basic Bacteriology Part III-1 (UPDATE) Basic Bacterial Genetics Dr Alaeddin Abuzant aladdin_abuzant@najah.edu

2. 2 Prokaryotic Gene Structure, Expression and Protein Secretion

3. 3 Bacterial Gene Structure The genetic information (the codons) of bacterial genes is normally continuous. That is to say, there are no introns in bacterial genes (in most cases) On the other hand, the genetic information of eukaryotic genes (codons that are found in the exons) are interrupted by non-coding regions/sequences known as introns.

4. 4 For a DNA region to be recognized as a gene it has to have: 1- Coding region/sequence (contains the codons) 2- Regulatory region(s): these are non coding sequence(s) such as the promoter to which RNA polymerase binds. Other regulatory DNA regions include enhancer and operator DNA regions, to which regulatory proteins (activator proteins and repressor proteins) bind to affect the activity of RNA polymerase positively or negatively, respectively.

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6. Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. 6 Gene expression: For a gene to be expressed, the gene has to be transcribed by RNA polymerase. This will generates mRNA which is then get translated by the ribosomes to generate a polypeptide. The generated polypeptide after that undergoes s folding and may be other post translation modification to give rise to a functional protein.

7. Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. 7 Transcription: Primary transcripts of eukaryotic gene contains exons and introns RNA processing: splicing to remove the introns. mRNA: ligation of exons (in addition 5 caping and poly A tailing)

8. 8 Alternative exons ligation: upon removal of introns from the primary transcript, the generated exons can be ligated in different arrangements so that more that one mRNA may be formed. Each of the formed mRNAs has a different sequence of the codons so that each of them can give a different protein upon translation. Accordingly: an eukaryotic gene may give rise to more than one mRNA and thus more than one protein

9. 9 Primary transcript of prokaryotic gene: Upon transcription of a prokaryotic gene, the generated RNA does not undergo processing because there are no introns. Accordingly, this trasncript can function as mRNA. Each prokaryotic gene give rise to one mRNA, and thus one protein is produced upon the translation of the prokaryotic mRNA

10. 10 In prokaryote, a gene (known also as operon) may have only one coding sequence or more than one coding sequence under the control of one promoter On the other hand, upon transcription of a prokaryotic gene, the generated mRNA does not undergo processing because there are no introns. Accordingly, each gene give rise to one mRNA, and thus one protein is produced upon the translation of mRNA Monocistronic operon; has one coding sequence  Upon transcription of monocistronic operon, the generated mRNA is called monocistronic mRNA because it has one the codons for one protein  Upon translation of the monocistronic mRNA only one protein is generated. Polycistronic operon: two or more coding sequences are found to be under the control of one promoter.  Upon transcription of polycistronic operon, the generated mRNA is called polycistronic mRNA since it contains codons for more than one proteins.  Upon translation of the polycistronic mRNA, (a big) polypeptide is generated that is cleaved to generate two or more proteins. In many cases, the generated proteins have related functions.

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12. 12 Note: In genetics, an operon is a functioning unit of genomic DNA containing a cluster of genes under the control of a single promoter. [1] The genes are transcribed together into an mRNA strand and either translated together in the cytoplasm, or undergo trans-splicing to create monocistronic mRNAs that are translated separately, i.e. several strands of mRNA that each encode a single gene product. The result of this is that the genes contained in the operon are either expressed together or not at all. Several genes must be co-transcribed to define an operon. [2]geneticsgenespromoter [1]transcribedmRNAtranslatedtrans-splicingmonocistronicexpressed [2] Originally, operons were thought to exist solely in prokaryotes, but since the discovery of the first operons in eukaryotes in the early 1990s, [3][4] more evidence has arisen to suggest they are more common than previously assumed. [5] In general, expression of prokaryotic operons leads to the generation of polycistronic mRNAs, while eukaryotic operons lead to monocistronic mRNAsprokaryoteseukaryotes [3][4] [5] http://en.wikipedia.org/wiki/Operon

13. 13 Polyribosomes Transcription and Translation Happen Simultaneously in Bacteria

14. Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. 14 Protein Maturation and Secretion To be functional as a protein, the generated polypeptide: –requires folding –association with other proteins and other compounds or metals –delivered to proper subcellular or extracellular site

15. Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. 15 Protein Translocation and Secretion Systems Numerous protein secretion pathways have been identified –some reside in all 3 domains –some unique to Bacteria and Archaea –some unique to gram-negative cells

16. 16 Protein Translocation and Secretion in Bacteria - 2 Translocation: is the movement of proteins from cytoplasm to plasma membrane or periplasmic space. Examples: include transport proteins, ETC proteins, proteins involved in chemotaxis and cell wall synthesis, enzymes. Two translocation system are known, the Sec translocation system and the Tat translocation system. Secretion : is movement of proteins from the cytoplasm to external environment Examples: hydrolytic enzymes for nutrient break down, toxins…… Six secretion systems have been recognized ( type I to Type VI)

17. Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. 17 Translocation Systems

18. Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. 18 Secretion Systems

19. Regulation of Gene Expression Prepared by Dr Alaeddin Abuzant, PhD Microbiology and Immunology Email: aladdin_abuzant@najah.edu

20. According to their expression: genes are classified into: 1- Constitutive genes ( house keeping genes): these genes are expressed all the time since their protein products are always needed 2- Regulated genes: are those genes that their expression is turned on and off depending whether these genes are needed or not at a particular situation

21. To say that a gene is being expressed, this implies that the following events have to happen: 1- Transcriptions: the gene is transcribed to mRNA by RNA polymerase 2- Translation: mRNA must be translated into a polypeptide 3- Post-translation events: The produced polypeptide undergoes folding, could be associated with other proteins, CHO or a metallic ion could be added or any other modification.

22. The expression of regulated genes can be controlled at different levels of gene expression: A- At the level of transcription 1- Transcription initiation 2- Transcription elongation B- At the level of translation 1- Translation initiation 2- Translation elongation C- At the level of post-translational modifications:

23. Regulated genes can be classified into: I- Inducible genes: most of the time, the expression of these genes is tuned of, but some times, their expression in needed to be turned on (Off→On). The expression of these genes usually happens upon the availability of a substrate that the bacterium can utilize, such as lactose. In this case, the product of these genes upon their expression is/are an enzyme or several enzymes that are involved in the degradation of this substrate. Accordingly, the gene product(s) of inducible genes are involved in a catabolic pathway.

24. II- Repressible genes: most of the time, the expression of these genes is tuned on, but sometimes, their expression is needed to be turned off (On→Off). Since the expression of these genes is ON most of the time, this implies that product of these genes is/are an enzyme(s) that are involved the biosynthesis of a compound that is needed most of the time such as an amino acid. Accordingly, these genes are involved in an anabolic pathway (biosynthetic pathway) When the produced compound accumulates and its amount becomes more than what is needed or when this compound in supplied to the culture medium in an excess amount, the expression of these genes is turned off.

25. The Expression Regulated Genes Is Controlled By: 1- Regulatory DNA regions such as operator site/region and activator site/region (Enhancer site) 2- Regulatory proteins that bind to these regulatory DNA regions (operator, activator) to affect the transcription level (increase or decrease transcription).

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27. Regulatory Proteins: Binding of these proteins to regulatory DNA regions either: 1- Activator proteins (activate transcriptions) (have a positive effect on transcription): these proteins are called activator proteins. These proteins usually binds to the Enhancer DNA region of a regulated gene 2- Repressor proteins that repress (stop/inhibit transcription) ( have a negative effect of transcription): these proteins are called repressors. These proteins usually binds to the operator DNA region of a regulated gene.

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30. 30 Regulatory Proteins can be found either in an Active or Inactive forms: As mentioned previously, repressor proteins ( repress/inhibit/stop) transcriptions through bonding to the operator region of a regulated gene. Active repressor: When ever the repressor protein is able to bind by itself to the operator region, this repressor is said to be active ( active repressor protein). To be removed/detach from the operator region, it needs a help. This can be seen in case of inducible genes (off most of the time) that is/are regulated an operator region and active repressor protein. Inactive repressor: In some cases, the repressor protein is NOT able to bind by itself to the operator region and it needs help to do so. This repressor protein is said to be inactive ( inactive repressor protein). This can be seen incase of repressible genes (On most of the time) if these genes are regulated by an operator region and Inactive repressor protein.

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32. 32 As mentioned previously, activator proteins (activate transcriptions) bind to the Enhancer DNA region of a regulated gene to promote or initiate trasncription. Active activator: When ever the activator protein is able to bind by itself to the enhancer region, this activator is said to be active ( active activator proteins). To be removed/detach from the enhancer, it needs a help. This can be seen in case of repressible genes (on most of the time) that is/are regulated an enhancer region (example, tryptophan operon) and an active activator protein.. Inactive activator: In some cases, the activator protein is NOT able to bind by itself to the enhancer region. It needs a help to be able to bind. This activator is said to be inactive ( inactive activator protein). This can be seen incase of inducible genes (off most of the time) if these genes are regulated by an enhancer region and inactive activator protein.

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34. 34 An example of Inducible geneses: Inducible genes usually encode for enzymes that are involved in catabolic pathways. In other words, these inducible are repressed most of the time (off most of the time) and are expressed only when their substrate (to be degraded or hydrolyzed) of these catabolic enzymes is available. This substrate (to be degraded or hydrolysed) is known as inducer Example of inducible genes: The lac operon: In the absence of lactose, the lac operon is repressed. Induction of lac operon transcription occurs only when lactose is available. One of the genes of this operon is β-galactosidase gene that encodes for β- galactosidase. This enzyme catalyzes lactose hydrolysis into galactose and glucose

35. Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. 35 Off → On

36. 36 Regulation of the lac Operon by the lac repressor (Lac I) Inducible Operon Lactose can be called as an inducer

37. 37 An example of repressible genes: Repressible genes usually encode for enzymes that are involved in anabolic pathways that synthesize a particular important substance for the cell. In other words, these repressible genes are expressed all the times (On most of the time) except when the concentration of the substance involved in its synthesis becomes more that what is needed. In this case, the high concentration of the produced substance has a negative impact of the expression of genes involved in its synthesis. Example of inducible genes: The tryptophan operon

38. Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. 38 On → Off

39. 39 Regulation of the trp Operon by Tryptophan and the trp Repressor Repressible Operon Tryptophan can be called as co-repressor

40. 40 Genes in bacteria are also called operon, Which could be: Monocitronic (one coding sequence under the control of one promoter Polycictronic: several coding regions that are regulated by one promoter

41. 41 Specialized Nomenclature Regulon: genes or operons controlled by a common regulatory protein Modulon: operons network under control of a common global regulatory protein but individual operons are controlled separately by their own regulators

42. Regulation of the Lactose Operon Expression (Lac Operon) (In Reality)

43. The Lactose Operon ( lac operon) : This operon is involved in lactose utilization. It is an inducible operon that only expressed when lactose is available to bacterial cells. This implies that most of the time, this operon is not expressed and it is only expressed when lactose is available. The Lac operon has three coding (lac z, lac y, & lac a) lac z encodes for Beta- Galactosidase that degrades lactose into glucose and agalactose lac y encodes for a Permeas or a lactose transporter lac a encodes for Transacetylase an enzymes involved in galactose utilization

44. Regulatory DNA regions of the lac operon includes: 1.A n enhancer region 2.A promoter 3.An operator Regulatory proteins involved in the regulation of the lac operon|: Lac I: which is a repressor ( an active repressor) that binds to the operator region spontaneously CAP: which is an inactive activator protein ( inactive activator) that binds to the enhancer region only when c-AMP is bound to it. Binding of cAMP to CAP activates it and makes it able to bind to the enhancer region Upon the presence of lactose, it binds to Lac I repressor causing lac I to leave the operator DNA region.

46. Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. 46 Note: In the absence of glucose, cAMP concentration is high so that cAMP binds to CAP and activates it and makes it able to bind to the enhancer region. Upon the presence of glucose, Glucose causes the concentration of cAMP to decrease, so that there will be no enough cAMP to bind to CAP. In this case, CAP will detach from the enhancer region. The effect of Glucose on the expression of the lac operon is called CATABOLIC REPRESSION

47. I- Both lactose and Glucose are absent When lactose is absent, lac I ( active repressor) binds to the operator site spontaneously When glucose is absent, the concentration of cAMP is high. So, it will bind to the CAP (inactive activator) and helps it to become active and thus to become able to bind to the enhancer region In this case CAP-cAMP binds to the Enhancer region BUT Lac I binds to the operator region CAP-cAMP activates transcription BUT Lac I suppresses transcription The net effect: NO TRANCRIPTION HAPPENS Expression of Lactose Operon is affected lactose and Glucose

49. I- Both lactose and Glucose are present The presence of glucose causes the concentration of c-AMP to decrease. In this case, the concentration of cAMP is low and thus c-AMP will NOT bind to CAP. CAP alone without C-AMP will detaches (leaves ) the enhancer region because by its self, CAP is an inactivate activator In the presence of Lactose, lactose (allolactose) binds to Lac I causing it to leave the operator region ( remember that lac-I alone is an active repressor, it binds sponatenously to the operator site. The binding of lactose to it will makes it inactive and thus it will detaches from the operator region The net effect: No activation of transcription occurs (CAP is not now bound to the enhancer region) No inhibition of transcription occurs (Lac I is not bound to the operator) Although these is no inhibition of transcription, there is no activation of transcription as well. Accordingly, the net effect will be NO TRANCRIPTION HAPPENS

51. III- Lactose is present but Glucose is absent In the absence of glucose, c-AMP concentration becomes high, thus, there will be enough cAMP to bind to CAP (CAP alone is an inactive activator and binding of cAMP to it will activates it). In this case, CAP-cAMP will binds to the enhancer region to activates transcription Recall that, lac I is an active repressor, so it means it binds to the operator by it self resulting in the inhibition of transcription. Since lactose is present, lactose will bind to Lac I, causing Lac I to become inactive and to leave the operator region. This means the transcription process is free to start Lactose is present but Glucose is absent means that: cAMP-CAP is bound to the Enhancer region------this will activate transcription Lac I is not bound to the operator -------- No inhibition of transcription Net effect: transcription will start

53. IV- Lactose is absent but Glucose is present In the presence of glucose, c-AMP concentration deceases. So, there will be no enough c- AMP to bind to CAP. Thus CAP will not be able alone (without cAMP, CAP in not active) to bind to the enhancer region. Thus no activation of transcription occurs. Recall that, lac I is an active repressor, so it means it binds to the operator DNA region by it self resulting in the inhibition of transcription. Since lactose is not present ( absent), Lac I will continue to bind to the operator region to inhibit the transcription process Lactose absent but Glucose is present means that: Lac I is bound to the operator resulting in inhibition of transcription since Lac I is a repressor CAP will NOT be bound to the enhancer region, thus, there will be no activation of the transcription process. Net effect: transcription will not start