mid 23 - Prokaryotic Transcription and Regulation Chapter 4...

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Unformatted text preview: Prokaryotic Transcription and Regulation Chapter 4, pp 106-130 Prokaryotic Transcription Central Dogma of Molecular Biology During transcription, the 4 base language of DNA is simply copied into the 4 base language of RNA. During protein synthesis, the 4 base language of RNA is translated into the 20-amino acid language of proteins. Copying of information transcription translation DNA RNA Protein One DNA strand acts as a template (read in the 3'5'), determining the order in which ribonucleoside triphosphate (rNTP) monomers are polymerized to form a complementary RNA strand. This polymerization reaction is catalyzed by RNA polymerase Transcription: RNA Synthesis Polymerization involves a nucleophilic attack by the 3' oxygen in the growing RNA chain on the phosphate if the next nucleotide to be added...resulting in a phosphodiester bond (energetically favorable). RNA synthesis: 5'3' Gene: a segment of DNA containing the information for a single polypeptide chain or functional RNA (i.e. rRNA, tRNA, etc.) The site at which RNA polymerase begins transcription is numbered +1. - Downstream: direction in which a template DNA strand is transcribed - Upstream: denotes the opposite direction - Nucleotide positions in the DNA sequence downstream are indicated by (+) and those downstream (-). Which DNA strand is the template strand? Upstream (-n) RNAP Promoter Basic Gene Structure +1 Downstream (+n) Gene 5' ACAT...ATG...TGA...ATGC 3' 3' TGTA...TAC...ACT...TACT 5' Terminator DNA Regulatory elements Transcribed region transcription 5' ACAU...AUG...UGA...AUGA 3' RNA Transcription Process: General View Initiation Elongation Termination "Closed Complex" "Open complex" (~14 bps) (Transcription initiation is considered complete when the first two ribonucleotides of an RNA chain are linked by a phosphodiester bond.) Transcription Process: General View Initiation Elongation Termination (primary transcript) A closer look at transcription termination: G/C rich A rich Intrinsic Terminators Terminator is usually a G/C-rich sequence followed by an A-rich sequence. An RNA:RNA stem loop forms (more stable due to G/C-rich in stem). This is followed by the formation of DNA:RNA hybrid via AU pairing. Release of RNA chain since A-U base pairing is less stable and is easily dissociated. A closer look at transcription termination: Rho-dependent rut T site DNA Rho binds RNA and moves along it using ATP RNA Pol pauses at T site T site Rho catches up Rho rut T site Rho is helicase-- unwinds RNA-DNA duplex Pol, mRNA, and Rho dissociates Transcription: Prokaryotes Structure of Prokaryotic RNA Polymerase Upstream DNA Downstream DNA Transcription in Prokaryotes Upstream Downstream The Holoenzyme: Direction of transcription Prokaryotic RNA pol is composed of 5 subunits: 22 complex = Core RNA polymerase Core pol + 70 factor = RNA Pol Holoenzyme 70 factor recognizes and binds to the promoter region Transcription in Prokaryotes Sigma factor (): subunit of RNA polymerase that recognizes and binds to the promoter + polymerase= holoenzyme 1. Holoenzyme is formed and factor interacts and binds to the promoter region Polymerase unwinds DNA Transcription begins After ~10 nt are synthesized, factor is released and polymerase undergoes conformational change. Elongation mode begins; RNA strand exits polymerase Elongation Polymerase encounters termination signal Full length RNA is released 2. 3. 4. 5. 6. 7. Simultaneous Transcription of a Gene by Multiple Molecules of RNA Polymerase Fine threads= newly synthesized transcripts Dots along DNA = RNA polymerase molecules What is the direction of transcription? Gene Structure: Cistron: an old name for a gene Polycistronic: 1 promoter directs synthesis of 1 mRNA that can be translated to more than one polypeptide Prokaryotic genes Polycistronic vs monocistronic Monocistronic: 1 promoter directs synthesis of 1 mRNA that translates to only 1 gene Eukaryotic genes Gene Structure: Polycistronic vs Ex. Synthesis of Tryptophan requires 5 enzymes in both prokaryotes and eukaryotes monocistronic The flow of genetic information is from DNA RNA protein. Transcription is the synthesis of RNA from DNA. Only genes are transcribed into RNA. Only mRNA is translated into protein. The basic building blocks for RNA are ribonucleoside triphosphates. RNA uses uracil (U) instead of thymine (T) in DNA. Transcription requires RNA polymerase activity. Prokaryotic transcription requires the factor to bind to the promoter region. Direction of RNA synthesis is 5' 3'. Unlike DNA, RNA is synthesized as single strands only. The process of transcription requires three general steps: initiation elongation termination. Summary Chapter 4 Control of Gene Expression in Prokaryotes Page 115-119 Transcriptional Regulation In multicellular organisms, specific cell types have differentiated to the point that they are highly specialized. Each cell type has arranged to express only some of its proteins. (Page 336 W) Bacteria also regulate the expression of their proteins. -Example: Synthesizing enzymes required to break down different carbon sources for energy (sugar metabolism). lactose glucose In an environment containing both glucose and lactose, E.coli cells preferentially metabolize glucose to make ATP. galactose maltose rhamnose Basic Control Units The cell must devise mechanisms to repress or shut down the transcription of all the genes encoding enzymes not needed and activate the transcription of those genes when the enzymes are needed. 1. 2. Cells need to be able to turn on or off the transcription of each specific gene or group of genes. Cells must be able to recognize environmental conditions in which they should activate or repress transcription of the relevant genes. Let's focus on lactose metabolism in E.coli: THE LAC OPERON! -The metabolism of lactose requires two enzymes: a) a permease to transport lactose in the cell b) -galactosidase to cleave the lactose molecule to yield glucose and galactose -glycosidic linkage -galactosidase H2O The Lac Operon Lac I CAP Lac promoter O lac Z lac Y lac A lacZ: -galactosidase, hydrolyzes lactose to glucose & galactose lacY: lactose permease, transports lactose into the cell lacA: transacetylase, removes other types of galactosides the cell does not use lacI: repressor protein, binds to the Operator (O) on DNA Coordinately Controlled Genes: Z, Y, A Remember... 1. 2. Cells need to be able to turn on or off the transcription of each specific gene or group of genes. Cells must be able to recognize environmental conditions in which they should activate or repress transcription of the relevant genes. When does E.coli want its lac operon genes expressed? When lactose is present in the absence of the more preferable glucose. The lac operon is controlled by two mechanims: 1) Negative control: The lac repressor 2) Positive control: CAP-cAMP complex 1) The Lac Repressor: Negative Control The lac repressor (LacI) is a molecule with two recognition sites: 1) One site that recognizes the specific operator sequence for the lac operon 2) Another distinct allosteric site that can bind lactose and certain analogs of lactose (IPTG). Allosteric site Y A Z LacI 2 scenarios: 1) In the absence of lactose, the repressor binds the operator region and blocks RNA polymerase from binding to the promoter. With no lactose present, the lac genes are not needed. The Lac operon: Negative Control -lactose X Lac I Lac promoter O lac Z lac Y lac A No mRNA transcription The Lac operon: Negative Control 2) When lactose is present it binds to the allosteric site of the repressor and causes a conformational change in the shape of the repressor's operator binding site. -After this conformational change, the repressor loses affinity for the operator and can no longer bind. RNA polymerase is now able to bind the promoter region and transcribe the Z, Y, and A. -Lactose (or derivatives) that inactivate the repressor and lead to expression of the lac genes are termed inducers. -Therefore, the lac operon is an example of an inducible operon. repressor lactose + lactose X Lac I Lac promoter O lac Z lac Y lac A Constitutive Mutants: LacI 1) One class of mutants synthesized all three enzymes at full levels, in the absence of an inducer. These constitutive mutants (always expressed in an unregulated fashion) were found to have mutations mapping close to but distinct from the Z, Y, and A genes. This led to the definition of the I locus as the region controlling the "inducibility" of the lac enzymes. Lac I Lac promoter O lac Z lac Y lac A I+ cells synthesize full levels of the lac enzymes only in the presence of an inducer. Whereas, I- cells synthesize full levels in the presence or absence of an inducer (constitutively active). 2) Another class of mutants constitutively expressed the lac enzymes even in the presence of an active repressor. Where do you think this mutation occurred? Constitutive Mutants: OC These constitutive mutants had mutations in the cis-acting operator region that did not allow binding of the repressor. These mutations are denoted OC (operator constitutive). With these nucleotide changes...the lac repressor no longer recognizes the operator site...leading to a constitutive activation of the lac genes. 2) Catabolite Control or Positive Control This second system of control exists because cells favor the uptake and metabolism of glucose over lactose. glucose > lactose Therefore, if both glucose and lactose are present, synthesis of galactosidase is not induced until all of the glucose has been utilized. Why does the cell do this? Catabolite Control or Positive Control Once glucose is depleted from the media, E.coli cells respond by synthesizing cyclic adenosine monophosphate (cAMP) cAMP Therefore...as [glucose] decreases [cAMP] increase Catabolite Control or Positive Control As the concentration of cAMP increases, it binds to a site in each subunit of a dimeric catabolite activator protein (CAP) protein, causing a conformational change that allows the protein to bind to the CAP site in the lac transcriptional control region. The DNA-bound cAMP/CAP complex interacts physically with RNA polymerase and increases the affinity of RNA polymerase for the lac promoter. + lactose -Glucose (high cAMP) Lac I CAP Lac promoter O lac Z lac Y lac A The Lac operon: Example of an inducible operon Lactose + lactose + glucose (low cAMP) lac Z lac Y lac A Lac I CAP Lac promoter O + lactose, + glucose: lactose binds to lac repressor and induces conformational change that inactivates the repressor Inactive lac repressor Pol-70 can proceed to transcribe Glucose metabolism is favored over lactose for ATP production. High glucose lowers cAMP accumulation, resulting in low or no CAP activity and low transcription transcription of lac operon. Summary Polycistronic mRNA Nucleotide sequence of binding sites in the control region of the lac operon cAMP-CAP binding site cAMP-CAP complex recognizes and binds to a specific site in the control region Binding of CAP causes conformational change, allowing RNA polymerase to transcribe operon The Lac operon: It all comes together High lactose, lack of glucose, get transcription of lac operon...-galactosidase -gal cleaves lactose into galactose & glucose (catabolites) As glucose levels increases, cAMP levels decreases and dissociates from CAP inactivating it. Lactose levels decreases and it dissociates from the lac repressor becomes active and binds to operator [galactose] [glucose] Biochemical Assay to assess gal Activity Galactose O-nitrophenol -gal activity can be assessed by the cleavage of ONPG (Orthonitrophenyl-b-D-galactopyranoside) ONPG is a derivative of lactose; it is a substrate for -galactosidase ONPG is a colorless compound When cleaved, it produces a yellow compound (O-nitrophenol) Trp operon: Example of repressible operon. Trp operon: Example of repressible operon. Repressible operon: An excess of product leads to a shutdown of the production of enzymes that synthesize that product. The repressor is unable to bind to the operator by itself...trp operon is transcribed...get tryptophan Repressor is only active when bound to a co-repressor, tryptophan The complex binds to the operator and suppresses transcription Feedback inhibition: In addition, tryptophan itself inhibits the first enzyme in the pathway (encoded by the trpE and trpD genes). Trp operon: Example of repressible operon. When tryptophan concentration is high, the operon is repressed, preventing overproduction of tryp Tryptophan concentration falls as it is being utilized Trp operon: Example of repressible operon. When trp concentration is low, most repressor molecules lack a co-repressor and fail to bind to the operator Tryp operon is transcribed to synthesize tryptophan How do bacteria senses change in the environment? Mechanisms that allows a cell to detect internal or external changes and confer appropriate responses are called signal transduction Bacterial cells often rely on the so-called two-component signal transduction system to detect environmental changes. Two components are: sensor: phosphorylated at a specific histidine residue in the "transmitter domain" in response to the environmental change. response regulator. "receiver domain" receives phosphate from sensor and becomes active Can function as activator or suppressor of genes PhoR-PhoB: two-component system Response to concentrations of free phosphate (Pi) in the environment Sensor: PhoR can bind Pi PhoR + Pi = inactive PhoR Pi = PhoR gets phosphorylated at H Phosphate then gets transferred to Asp of PhoB, response regulator Becomes active trxn factor to turn on genes needed to cope with low Pi environment Prokaryotic Translation Translation Translation is the whole process by which the nucleotide sequence of mRNA is used to order and to join the amino acids in a polypeptide chain. In prokaryotes, translation occurs in the same cytoplasm as transcription. 3 types of RNA molecules are essential for translation to occur: 1) Messenger RNA (mRNA): carries genetic information transcribed from DNA in the form of a series of 3-nucleotide sequences, called codons, each of which specifies a particular amino acid. 2) Transfer RNA (tRNA): small RNA chain (74-93 nucleotides) that is key to deciphering the codons in mRNA. Each amino acid contains its own subset of tRNAs. The correct tRNA with its attached amino acid is selected at each step (Each tRNA molecule contains a three-nucleotide sequence, an anticodon, that can base-pair with its complementary codon in the mRNA. The Three Roles of RNA in Translation 3) Ribosomal RNA (rRNA): associates with a set of proteins to form ribosomes that composed of a large and small subunit. Ribosomes bind tRNAs associated with amino acids and physically move along an mRNA molecule, catalyzing the assembly of amino acids into polypeptide chains. The Genetic Code Genetic information stored in DNAgets transcribed into RNAthe messenger Message mRNA has to be decoded into protein...Use the "genetic code." The genetic code used by cells is a triplet code, which consists of a three nucleotide sequence or codon. The nucleotide sequence representing individual amino acids is called codons The sequence complementary to the codon is the anti-codon. The genetic code is universal (with some exceptions...animal mitochondria) A codon is a 3-letter code (nucleotide triplets); each codon specifies an amino acid or one of three stop signals. Why a 3-letter code? There are 4 different bases per position: -If 2 letter code (42), then have 16 codons...not enough for 20 aa -If 3 letter code (43), the have 64 codons...more than enough -If 4 letter code (44), then have 256 codons...too much The Genetic Code is Degenerate Conclusion: The DNA code is degenerate (there is more than one triplet code for each aa) Deciphering the Genetic Code 1.All 64 codons are used: 61 of them can be assigned to certain amino acids, the other three are stop signals. One of the codons can act both as an amino acid codon and as a start signal. 2.The different amino acids have different numbers of accompanying codons. For some, like Met or Trp exists just one codon, for others two or four and for some (Leu, Ser, Arg) even six. The frequency of the codons and the frequency of their amino acid is correlated. An exception is Arg, that has six codons but is underrated regarding its frequency in proteins. 3.The codons are not assigned randomly. The first two nucleotides of a codon have a higher informational value than the third one, GUU, GUC, GUA and GUG, for example, do all encode Val. Many (nearly 30%) of all base substitutions do not change the encoding properties, for example: UUU > UUC: Phe > Phe Genetic Code: Where to begin and stop? Synthesis of all polypeptide chains in prokaryotic and eukaryotic cells begins with the amino acid methionine. -In most mRNAs, the start (initiator) codon specifying this amino-terminal methionine is AUG. -Exceptions: GUG in prokaryotes CUG in eukaryotes The three codons UAA, UGA, and UAG do not specify amino acids but constitute stop (termination) codons that mark the carboxyl terminus of polypeptide chains. The sequence of codons that runs from a specific start codon to a stop codon is called a reading frame. -This precise linear array of ribonucleotides in groups of three specifies the precise linear sequence of amino acids in a polypeptide chain. The Genetic Code Is Non-Overlapping G G G The Genetic Code Is Non-Overlapping Sequence original If overlapping then... If non-overlapping then... AGCATCG single-base substitution AGC, GCA, CAT, ATC, TCG AGC, ATC, G.... AGAATCG AGA, GAA, AAT, ATC, TCG AGA, ATC, G... Conclusion: as seen from studies of mutant proteins, such as hemoglobin from sickle cell anemia, the mutant form contains ONLY ONE amino acid substitution Therefore the genetic code is non-overlapping because each nucleotide is part of only one codon, resulting in only one amino acid replacement Genetic Code and Reading Frames Depending on where you start, any nucleotide sequence can be read as three different "reading frames." An open reading frame (ORF) is the nucleotide sequence between a start codon and a stop codon. Except for the ORF, the other 2 reading frames usually contain many stop codons. Effect of Mutation on Gene Structure and Expression Frameshifts: insertions or deletions of bases not in multiples of 3 will result in a frameshift. The entire a.a. sequence following the mutation will differ from the original sequence. Deletion X Decoding the information in mRNA: tRNA tRNA is part of the decoder All tRNAs are about the same length (~ 80 nt long) & structure Contains 3 loops; one of the loop contains an anticodon region that complements the codon in mRNA. An amino acid is covalently attached to the acceptor stem at the 3' tail of CCA. 2D structure of tRNA ...
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