2 INTRODUCTION TO MACROMOLECULAR SYNTHESIS 2INTRODUCTION TO MACROMOLECULAR SYNTHESISAlthough there are exceptions, control of metabolic processes usually occurs at the initiation stage.A. ReplicationReplication of the chromosome is tightly regulated such that replication is begun once and only once per cell cycle and at the right time in the cell cycle. This regulation is exerted by a regulatory protein or proteins acting at the replication origin to control the initiation stage. Once DNA synthesis has begun, it normally proceeds to complete duplication of the entire chromosome.B. Gene ExpressionGene expression generally means all the processes necessary to produce a functional product. That is, both transcription and translation (except for genes which do not produce protein products, such as tRNA and rRNA genes, in which case gene expression would involve transcription and processing of the RNA products). Gene expression also involves protein (often enzymes) function. Gene expression also is tightly regulated, usually by regulating transcription. Transcription usually is regulated by controlling the initiation of transcription. Genes are subject to negative, positive, or negative and positive controllers. (Some genes are not regulated; they are transcribed at a constant, usually low, level and are said to be expressed constitutively.)Negative control is exerted by repressors which bind operators and inhibit initiation of transcription. Repressors can be either active or inactive, depending on the presence or absence of low molecular weight compounds in the environment (or other environmental factors).Positive control is exerted by activator proteins which bind activator binding sites near promoters and stimulate initiation of transcription. Activator proteins can be either active or inactive, depending on the presence or absence of low molecular weight compounds in the environment (or other environmental factors).Repressors and activators act together to turn genes or operons on or off. They form a molecular switch. These switches function to turn genes on or off over one to two generations. That is, about thirty minutes to a couple of hours can be required for this switch to change completely from on to off or off to on.Fine tuning of gene expression is done by the process of attenuation. That is, if a gene or operon is turned on, the level of expression can be adjusted within minutes to produce more or less product. A gene or operon which is being transcribed can stop transcription momentarily near the promoter (that is, after polymerization of about 200 nucleotides) if the entire messenger is not needed. This is economical because it saves the polymerization of several thousand nucleotides in the complete messenger, at the cost of several hundred nucleotides at the 5' end. Attenuation depends on a signal to terminate transcription early; this signal depends on the intracellular concentration of some low molecular weight compound. An example is attenuation of an amino acid biosynthesis operon. The intracellular concentration of the amino acid acts as a signal for the growing mRNA to form a terminator stem:loop near its 5' end; this terminates the synthesis of messenger early. Attenuation is another form of molecular switch.
3 GENE EXPRESSION – ALL EVENTS NECESSARY FOR GENE PRODUCT TO FUNCTION (USUALLY CATALYZE REACTION):TRANSCRIPTION, TRANSLATION, PROTEIN ACTIONCONTROL OF GENE EXPRESSION IS MOSTLY AT WHAT LEVEL?TRANSCRIPTION, TRANSLATION, PROTEIN ACTION?TRANSCRIPTION – OF COURSECONTROL OF TRANSCRIPTION IS MOSTLY AT WHAT LEVEL?INITIATION, POLYMERIZATION, STABILITY OF mRNA?INITIATION – OF COURSEWHAT DETERMINES THE FINAL AMOUNT OF GENE PRODUCT?AMOUNT OF mRNA SYNTHESIZED – OF COURSE
4 OPERON PRODUCTS INVOLVED IN CATABOLISM – GOAL OF CELL IS TO SYNTHESIZE AS MUCH ENZYMES (CODED BYTHAT OPERON) AS POSSIBLE TO BRING IN FOOD, OXIDIZE IT,UTILIZE IT AND GROW AS RAPIDLY AS POSSIBLE.LAC OPERON – LAC ENZYMES FUNCTION FOR CELLSTO USE LACTOSE AS FOOD.OPERON PRODUCTS INVOLVED IN ANABOLISM – GOAL OFTHAT OPERON) AS NEEDED TO SYNTHESIZE SOME METABO-LITE IN THE AMOUNT NEEDED FOR GROWTH.TRYPTOPHAN OPERON – TRYP ENZYMES SYNTHESIZETHE AMINO ACID TRYPTOPHAN.
5 GENE EXPRESSION CONTROL 3GENE EXPRESSION CONTROLOPERON STRUCTURESites on DNA where proteins bind - Promoter, Operator, Activator Binding SitesStructural genes which encode proteins (e.g., enzymes)Some genes encode stable RNA productsCistron, polycistronic mRNARepressor, Negative controlActivator, Positive controlLACTOSE OPERON (catabolic)Lactose (glucose, galactose, beta galactose, beta galactosidase)Lactose operon genes -Beta galactosidase, permeaseControl Sites (promoter, operator, activator binding site)Repressor gene, repressor, active repressor, inducer, inactive repressorPositive control, cAMP, Catabolite Activator ProteinTRYPTOPHAN OPERON (anabolic)Structural genesControl sites (promoter, operator, attenuation site)Repressor - inactive- co-repressor, active repressorAttenuationcoupled transcription and translationfine tuner of gene expressionleader peptide, trp codonsalternative structures of mRNA, molecular switch
6 BACTERIAL GENES ARE SUBJECT TO: 4BACTERIAL GENES ARE SUBJECT TO:NEGATIVE CONTROL REPRESSOR PROTEIN - ACTIVE/INACTIVEPOSITIVE CONTROL ACTIVATOR PROTEIN - ACTIVE/INACTIVEBOTH POSITIVE AND NEGATIVE CONTROL FINAL RESULT DEPENDS ON INTERACTION OF BOTH MECHANISMSNEITHER POSITIVE NOR NEGATIVE ALWAYS TRANSCRIBEDUSUALLY AT LOW LEVEL-CONSTITUTIVE-
7 TRANSCRIPTION CONTROL (INITIATION) REPRESSOR CONTROLS NEGATIVELY 5TRANSCRIPTION CONTROL (INITIATION)REPRESSOR CONTROLS NEGATIVELY3'5'3'REPRESSORGENEREPRESSORmRNA5'3'ACTIVE/INACTIVEDEPENDS ONENVIRONMENTREPRESSOR
8 LITTLE OR NO TRANSCRIPTION CISTRON; POLYCISTRONIC mRNA 6ACTIVE REPRESSOR - BINDS OPERATORINHIBITS TRANSCRIPTION [INITIATION]LITTLE OR NO TRANSCRIPTIONINACTIVE REPRESSOR - CANNOT BIND OPERATORRNA POLYMERASE TRANSCRIBESRNA POLYMERASEOPERON; OPERATORCISTRON; POLYCISTRONIC mRNA
9 ACTIVATOR PROTEINS CONTROL POSITIVELY 7ACTIVATOR PROTEINS CONTROL POSITIVELYACTIVATORBINDINGSITE5'5'3'PROMOTERACTIVATORPROTEINGENE5'3'ACTIVATORmRNAACTIVE/INACTIVEDEPENDS ONENVIRONMENTACTIVATOR
14 LACTOSE GENES (OPERON) 12LACTOSE GENES (OPERON)PROMOTERCAP SITEREPRESSORGENEOPERATORCAP = CATABOLITEACTIVATOR PROTEINZ = b - GALACTOSIDASEY = LACTOSE TRANSPORTREPRESSORPROTEIN (ACTIVE)NO LACTOSE (NO INDUCER)ACTIVE REPRESSOR BINDS OPERATORLITTLE OR NO TRANSCRIPTION~10 MOLECULES b - GLACTOSIDASE/CELLLACTOSE MEDIUM (INDUCER)INDUCER BINDS REPRESSORREPRESSOR INACTIVATEDCANNOT BIND OPERATORTRANSCRIPTION PERMITTED> 103 MOLECULES b - GALACTOSIDASE/CELLNOBELJACOB, MONOD, LLOWF
15 LACTOSE OPERON IS SUBJECT TO POSITIVE CONTROL 13LACTOSE OPERON IS SUBJECT TO POSITIVE CONTROLGROW IN LIMITING CONCENTRATIONS OF GLUCOSE AND LACTOSELOG TURBIDITYTIME
16 LACTOSE OPERON IS SUBJECT TO POSITIVE CONTROL 14LACTOSE OPERON IS SUBJECT TO POSITIVE CONTROLGROW IN LIMITING CONCENTRATIONS OF GLUCOSE AND LACTOSEb - GALACTOSIDASE LEVELLOG TURBIDITYTIMECELLS USE GLUCOSE FIRSTNO b - GALACTOSIDASE IN PRESENCE OF GLUCOSEGLUCOSE PREVENTS POSITIVE CONTROL FROM ACTIVATINGGLUCOSE INHIBITS cAMP SYNTHESIScAMP = CYCLIC ADENOSINE MONOPHOSPHATE - NEEDED TO ACTIVATE CAPCAP = CATABOLITE ACTIVATOR PROTEIN
17 POSITIVE CONTROL OF LAC 15POSITIVE CONTROL OF LACcAMP BINDS CAP; ACTIVATES CAPcAMP-CAP BIND CAP SITE TO STIMULATE TRANSCRIPTIONATPGLUCOSEINHIBITSADENYLATE CYCLASE+ PPiCYCLIC AMP
18 MAXIMUM LAC TRANSCRIPTION 16MAXIMUM LAC TRANSCRIPTION+1CAP SITEPROMOTEROPERATORREPRESSOR AND INDUCER ISINACTIVE - CANNOT BIND OPERATORCAPGENECATABOLITE ACTIVATOR PROTEINCAP BINDS cAMP, BECOMES ACTIVECAP cAMP BIND CAP SITE- STIMULATERNA POLYMERASEPOSITIVE AND NEGATIVE CONTROL SYSTEMS ALLOW CELLSTO USE MORE EFFICIENT CARBON/ENERGY SOURCE FIRSTTHAT IS: CHOOSE BETWEEN CARBON SOURCES
19 MAXIMUM LAC EXPRESSION REQUIRES: 17MAXIMUM LAC EXPRESSION REQUIRES:INDUCER - TO INACTIVATE REPRESSOR [INDUCTION]ANDPOSITIVE ACTIVATIONcAMP CAP [CATABOLITE ACTIVATOR PROTEIN]MUST BIND CAP SITE TO STIMULATE TRANSCRIPTION
20 18FOR CELLS GROWING IN THE FOLLOWING CONDITIONS, THE LAC OPERON WILL BE CONTROLLED IN THE FOLLOWING WAYS:GROWTH MEDIUMNEGATIVE CONTROL REPRESSOR-OPERATORPOSITIVE CONTROL cAMP-CAP ACTIVATORRESULTGLUCOSE (ONLY)LAC REPRESSOR IS SYNTHESIZED;IS ACTIVE;BINDS OPERATOR;INHIBITS RNA POLYMERASEGLUCOSE INHIBITS ADENYLATE CYCLASE;NO cAMP SYNTHESIS;cAMP-CAP CANNOT BIND ACTIVATOR SITELAC IS OFFLACTOSE (ONLY)IS INACTIVATED BY LACTOSE; (ALLO-LACTOSE);CANNOT REPRESScAMP IS SYNTHESIZED;cAMP AND CAP BIND ACTIVATOR SITE;STIMULATE RNA POLY-MERASE TO TRANSCRIBELAC IS ONGLUCOSE AND LACTOSELAC IS OFF (UNTIL ALL THE GLUCOSE IS USED)