Trp operon

GENE REGULATION - TRP OPERON

1. GENE REGULATION IN PROKARYOTES S.PALANIANANTH 1 ST MSC MICROBIOLOGY AYYA NADAR JANAKI AMMAL COLLEGE SIVAKASI

2. TRP OPERON SUBMITTED TO : DR. S. MUNIASAMY ASSISTANT PROFESSOR OF MICROBIOLOGY AYYA NADAR JANAKI AMMAL COLLEGE SIVAKASI

3. • Gene regulation Gene regulation : It is the process by which information from a gene is used in the synthesis of a functional gene product . These products are often proteins, but in non-protein coding genes such as rRNA genes or tRNA genes, the product is a functional RNA . Types of regulation of gene expression Positive regulation or induction : When the expression of genetic is quantitatively increased by the presence of specific regulatory element is known as positive regulation. Element modulating positive regulation is known as inducer, activator or positive regulator . Negative regulation or repression. When the expression of genetic information diminished by the presence of specific regulatory element . The element or molecule mediating the negative regulation is said to be repressor Activate

4. Generalized structure of operon Promoter: A promoter is a nucleotide sequence in the DNA that initiates transcription of a particular gene. Operators : These are segments of DNA that regulate the activity of the structural genes of the operon .• Structural Genes: A gene that codes for any RNA or protein product other than a regulatory factor • Regulator These nucleotide sequences control the operator gene in cooperation with certain compounds called inducers and co-repressors present in the cytoplasm .

5. Operon The operon, principle of gene expression An operon is a functioning unit of genomic DNA containing a cluster of genes under the control of a single promoter . In bacteria, genes that encode for proteins with closely related functions are found grouped along with cis-acting regulatory elements that determine the transcription of these genes, thus these genes are regulated in a coordinated way. These clusters of genes are called operons. The transcription product of an operon is a single polycistronic MiRNA T ypes of operon Inducible operons : They include genes that encode for enzymes that take part in metabolic pathways.The expression of the gene is controlled by the substrate. Example is the " Lac Operon“ Repressible operons : They include genes that encode for enzymes involved in biosynthetic pathways.The expression of the gene is controlled by the end-product of the pathway. Example is the " Trp Operon".

6. INTRODUCTION The trp operon is an operon a group of genes that is used , or transcribed that codes for the components for production of tryptophan. The trp operon is present in many bacteria but was first characterized in E.coli. Trp operon discovered in 1953 by Jacques Monod and colleagues , the trp operon in E.coli was the first repressible operon to be discovered. Tryptophan is an aromatic amino acid , and is required for synthesis of all protein that contain tryptophan. If tryptophan is not present in the medium in adequate quantity, the bacterial cell has to make it, as it is required for the growth of the bacteria .  The tryptophan operon, found in E.coli bacteria, it is a group of genes that encode biosynthetic enzymes for the amino acid tryptophan ( Repressible OPERON ) The tryptophan operon is expressed (turned on) when tryptophan levels are low and repressed (turned off) when they are high

7. INTRODUCTION The  trp  operon is regulated by the  trp  repressor. When bound to tryptophan, the  trp  repressor blocks expression of the operon. Tryptophan biosynthesis is also regulated by   attenuation  (a mechanism based on coupling of transcription and translation).

8. What is the  trp  operon? Bacteria such as  Escherichia coli  (a friendly inhabitant of our gut) need amino acids to survive —because, like us, they need to build proteins. One of the amino acids they need is tryptophan. If tryptophan is available in the environment,  E. coli  will take it up and use it to build proteins. However,  E. coli  can also make their own tryptophan using enzymes that are encoded by five genes. These five genes are located next to each other in what is called the  trp  operon. If tryptophan is present in the environment, then  E. coli   bacteria don't need to synthesize it, so transcription of the genes in the  trp  operon is switched "off." When tryptophan availability is low, on the other hand, the operon is switched "on," the genes are transcribed, biosynthetic enzymes are made, and more tryptophan is produced.

9. Structure of the  trp  operon

10. Structure of the  trp  operon Tryptophan operon consists of  structural gene  and   regulatory gene. I . Regulatory gene  are Promoter, Repressor, Operater and Leader sequence. II. Structural gene   are TrpE , TrpD , TrpC , TrpB and TrpA trpE : It enodes the enzyme Anthranilate synthase I trpD : It encodes the enzyme Anthranilate synthase II trpC : It encodes the enzyme N-5’-Phosphoribosyl anthranilate isomerase and Indole-3- glycerolphosphate synthase trpB : It encodes the enzyme tryptophan synthase-B sub unit trpA : It encode the enzyme tryptophan synthase-A sub unit  

11. Turning the operon "on" and "off" What does the operator do?  This stretch of DNA is recognized by a regulatory protein known as the   trp  repressor . When the repressor binds to the DNA of the operator, it keeps the operon from being transcribed by physically getting in the way of RNA polymerase, the transcription enzyme . The   trp  repressor does not always bind to DNA. Instead, it binds and blocks transcription only when tryptophan is present. When tryptophan is around, it attaches to the repressor molecules and changes their shape so they become active. A small molecule like tryptophan , which switches a repressor into its active state, is called a  corepressor .

12. Tryptophan operon is regulated by following mechanism 1. Repression When there is little tryptophan in the cell , on the other hand, the  trp  repressor is inactive (because no tryptophan is available to bind to and activate it) . It does not attach to the DNA or block transcription , and this allows the  trp  operon to be transcribed by RNA polymerase . In this system, the  trp  repressor acts as both a sensor and a switch . It senses whether tryptophan is already present at high levels, and if so, it switches the operon to the "off" position, preventing unnecessary biosynthetic enzymes from being made.

13. Repression When tryptophan is high in cell then it binds with repressor protein and change its confirmation so that it become active and bind to the operator near promoter. Binding of repressor protein to operator overlaps the promoter, so RNA polymerase cannot bind to the promoter. Hence transcription is halted. Since tryptophan is already high in cell, no transcription of structural gene is required for biosynthesis of tryptophan. This is also known as negative regulation.

14. High tryptophan

15. Tryptophan operon is regulated by following mechanism 2 . Attenuation: In bacteria , transcription and translation occurs simultaneously . The translation starts before transcription completes. In this attenuation mechanism, rate of translation determines whether transcription continues or terminates . Therefore the attenuation mechanism is only found in bacteria but not in eukaryotic cell . It Involves the trpL gene , a sequence of 162 nucleotides that produces an mRNA leader sequence; near the 3’ end of this leader sequence ( trpL gene) is a region called the attenuator . .

16. Leader sequence ( trpL ) play important role in attenuation. Leader sequence contains such a nucleotide sequence that mRNA transcribed from it contains four specific region. Region 1, region 2, region 3 and Region 4. Region 3 is complementary to both region 2 and region 4. If region 3 and region 4 base pair with each other, they form a loop like structure called attenuator and it function as transcriptional termination . If pairing occur between region 3 and region 2, then no such attenuator form so that transcription continues. Region 1 is the most important region that determines whether to form loop between region 2-region 3 or region 3-region 4. The region 1 consists of sequence of 14 codons, out of which two codons are tryptophan codon (codon 10 and 11)

17. When tryptophan is high in cel l than tRNA carrying tryptophan encodes codon 10 and 11 . Such that ribosome encloses the region 2 which is near to the tryptophan codon. Hence region 3 base pair with region 4 to form attenuator as region 2 is not available for pairing. Consequently, transcription is halted . When tryptophan is low or absent in cell, then translation stops at the position of tryptophan codon. Such that loop between region 2 and region 3 forms. Transcription continues.

18. The biosynthesis of tryptophan

19. The biosynthesis of tryptophan TRP E-Anthranilate synthetase I Converts chorismate to anthranilate using ammonia. Anthranilate is an amino acid that is later broken down into another amino acid, Trysoine. TRP D-Anthranilate synthetase IIA type of Glycosyltransferase that participates in the synthesis of Tryptophan Produces an amino acid called Anthranilate which ,by the help of TRP E, is broken down into another amino acid, Trysoine. Converts -(5- phospho -D- ribosyl )-anthranilate and diphosphate to anthranilate and 5- phospho -alpha-D-ribose 1-diphosphate. It is 1 of the 2 components that together encode Anthranilate synthetase

20. TRP C- indole-3-glycerol-phosphate synthase Involved in the synthesis of tryptophan and amino acids. It is a type of lyase that cleaves carbon-carbon bond. this ability allows it to converts 1-(2- carboxyphenylamino )-1- deoxy -D-ribulose 5-phosphate to C(1)-(3- indolyl )-glycerol 3-phosphate and carbon dioxide and water TRP B tryptophan synthase subunit B There are two beta subunits which form a dimer Beta dimer catalyzes the formation of L-tryptophan from L-serine and 1-( indol-3- yl )glycerol 3-phosphate

21. TRP A tryptophan synthase subunit A Tryptophan synthase is an enzyme found in plants and bacteria There are two separate alpha subunits They catalyze the formation of indole and glyceraldehyde 3-phosphate from indole glycerol phosphate in tryptophan biosynthesis, which is needed by the B subunits .

22. Questions What is meant by catabolism and anabolism process? TRP OPERON IS ANABOLIC OR CATABOLIC Role of RNA polymerase? Define Apo enzyme? TRP OPERON OR OTHERWISE CALLED? ARE OPERONS PRESENT IN EUKARYOTES? WHEN WILL TRP OPERON IS TURNED ON? AN TURNED OFF? WHAT IS POLYCISTRONIC MRNA? HOW MANY STRUCTURAL GENES ARE PRESENT IN THE TRP OPERON ? TRP OPERON IS AN EXAMPLE OF ______________________