Gene expression Flashcards
Regulating gene expression in both bacteria and eukaryotes
Compactness of DNA - difficult to transcribe very compact DNA
Alteration of structure - if and where DNA is opened up, how open it is
Transcription - production of pre-mRNA is regulated through whether or not gene is transcribed
mRNA processing - where and how pre-mRNA is processed (capping with 7-methylguanosine, addition of polyA tail, removal of introns), does not apply to prokaryotes
Translation - regulation of whether mRNA is translated or not, timing of translation
Post-translational modifications - addition of groups to change function and activity of protein
Constitutively expressed genes
certain gene products are essential components of living cells and are continually expressed in most cells
Inducible and repressible genes
Some gene products are only expressed under certain environmental conditions.
e.g
genes involved in using lactose as a carbon source are inducible
Glucose is preferred carbon source in bacteria but cells can adapt if glucose is absent
Gene expression is induced when lactose is present and glucose is absent
Bacterial cell induces production of lactose-metabolizing enzymes which break down lactose into glucose and galactose, glucose is now available for cell to use
Catabolic pathways such as the one described are often inducible
E.g. when tryptophan is present, certain genes are repressed
Bacterial cells synthesize their own tryptophan but when Trp is present, they have no need to make their own so those biosynthetic genes are repressed
Repression occurs at level of transcription of Trp biosynthetic genes
Anabolic pathways are often repressible (no enzymes synthed for breakdown)
Bacterial operons
An operon is a group of genes that are involved in some function such as tryptophan biosynth or lactose metabolism; a transcriptional unit that describes linked series of functional units in gene
Operon expression is controlled by a single promoter which is responsible for transcribing all of the genes into RNA which is later translated to separate proteins which form the biosynthetic pathway
Coordinate expression - expression of structural genes controlled as a single unit (operons)
Operator - DNA sequence that binds a regulatory protein that starts/stops in transcription of operon
Operons can control bacterial gene expression in a few different ways:
Negative inducible operons - e.g. lac operon, occur with or without an inducer
No inducer present - no lactose present, operon is not expressed (lactose = inducer)
In absence of lactose, a regulatory/repressor protein (separate gene) will bind to the operator of the lac operon and block RNA polymerase from synthesizing mRNA that codes for lactose metabolizing enzymes
When lactose is present, repressor protein binds lactose and is inactivated, so RNA polymerase can access promoter and start transcribing genes for lactose enzymes
Negative repressible operons - not described in this course
When Trp is present, it binds to repressor protein so protein blocks RNA polymerase
Positive control - regulatory protein is an activator which binds to DNA and interacts with RNA polymerase to assist efficiency of transcription of structural genes
NOTE: the lac operon of E. coli is a negatively inducible but positively regulated operon
Controlled by two factors: presence of lactose and glucose
LAC OPERONS IMPORTANT
Genes of lac operon are transcribed ONLY when lactose is present and glucose is absent
Lac operon is negatively inducible and located between the purE and proA operons
Lac repressor is encoded by the I gene and is separate from the lac gene
Lac operon consists of a promoter, an operator, and 3 structural genes: Z, Y, and A genes (see diagram)
Lactose permease (Y gene) - lactose transport into cell
β-galactosidase (Z gene) - main purpose is to cleave lactose into glucose and galactose
Low background level converts lactose into allolactose, the lac operon inducer
Lac I region - encodes lac repressor, a diffusible tetramer protein that represses lac operon in absence of allolactose (I gene mentioned above)
Lac O region - lac operator, regulator DNA sequence that works to control lac operon in cis
Sequences that work in cis only affect genes that are downstream from them
Lac repressor binds to lac operator in absence of allolactose inducer
Lac O overlaps the initiation start site so when repressor is bound, RNA polymerase cannot access the promoter, no lac operon transcription occurs
Lac operon expression is OFF when allolactose inducer is absent
Glucose is present, no lactose metabolizing enzymes synthesized because repressor is bound to operator and RNA polymerase cannot transcribe the gene
Lac operon expression is ON when allolactose inducer is present
Glucose is absent, lactose is present, and metabolizing enzymes are synthesized because allolactose inducer causes the repressor to release from the operon and RNA polymerase can transcribe the gene
Mutations of wild type lac operon (I+P+O+Z+Y+A+, allolactose inducible for all lac operon enzymes):
I+P+O+Z+Y -A- - allolactose inducible only for lacZ, would only make functional protein for lacZ
Mutations in the I gene (I-) or the operator sequence (OC) result in constitutive expression of downstream wild-type lac Z, Y, and A genes
OC mutations act only in cis on the downstream Z, Y, and A genes
Merozygote - partial diploid bacterial cell, e.g. E. coli with lac genes in chromosome and in F’ factors (conjugative plasmids)
F’ I+P+OCZ+Y+ (F’ factor) / I+P+O+Z-Y -A- (chromosome) - constitutive expression of lacZ from the F’ factor (since the chromosome lacks the wildtype Z gene)
F’ I -P+O+Z-Y - / I+P+OCZ+Y +A+ - constitutive expression of lacZ, Y, A from chromosome
F’ I -P+O+Z-Y - / I -P+O+Z+Y +A+ - constitutive expression of lacZ, Y, A from chromosome
No expression from F’ factor because Z and Y are mutant
NOTE: wild type Z+Y +A+ alleles are dominant to the Z-Y -A- alleles (because mutant produces no functional enzymes), I+ is dominant to I -
All wild-type F’ / mutant Z, Y, A chromosome - allolactose inducible for lac Z, Y, A on F’
Opposite of above - allolactose inducible for lac Z, Y, A on the chromosome
Wildtype for both but mutant repressor (I-) on chromosome - allolactose inducible for both F’ and chromosome.
Repressor is diffusible so it can bind operator on same DNA molecule or another one
That means that despite the fact that wild type is dominant to mutant, the repressor protein can still function because it is diffusible
EXAM QUESTION - identify phenotype from genotype of lac operon
Question might not write whole genotype, may only indicate mutants
LacI super repressors (lacIS) - in absence of allolactose, wild type lac repressor can bind to operator and prevent transcription of lac enzymes. In presence of allolactose, wild type repressor is inactivated which permits expression of lac enzymes
Some lacI mutants cannot be inactivated by allolactose because the inducer binding site is altered, meaning the lacI repressor is always bound to the operator, preventing transcription
Other lac mutations include promoter mutations (denoted lacP -) which interfere with binding of RNA polymerase to the promoter, thus preventing lac operon transcription
Lac operon is not necessarily always induced in the presence of allolactose - glucose must be present
Glucose is preferred over lactose as the carbon source, prevents lac operon induction through negative regulation, aka catabolite repression
Glucose regulated lac operon indirectly through its effects on cyclic AMP (cAMP) levels
cAMP is a signal molecule, derivative of ATP
When glucose is high, cAMP levels are low and vice versa
cAMP levels regulate lac operon activity - high cAMP levels cause cAMP to bind another protein called the catabolite activator protein (CAP)
Interaction of cAMP with CAP permits binding to a special region in lac promoter that promotes binding of RNA polymerase (to start lac operon transcription)
cAMP/CAP complex promoter binding is REQUIRED for lac operon transcription
cAMP/CAP helps orient RNA polymerase and associated σ factor to start transcription
