GENE REGULATION IN PROKARYOTES

7TH WEEK, BIO-1053 GENE REGULATION IN PROKARYOTES 7th week General Genetics-BIO1053 7th week General Genetics-BIO1053 Histochemical localization of GUS expression under control of the Atndt1 and Atndt2 promoter in A thaliana 7th week General Genetics-BIO1053 Hierarchical clustering of top-500 most variable genes 7th week General Genetics-BIO1053 Chapter outline Overview of Prokaryotic Gene Regulation The Regulation of Gene Transcription Attenuation of Gene Expression: Termination of Transcription Global Regulatory Mechanisms: E.coli’s response to heat shock 7th week General Genetics-BIO1053 The regulation of gene expression can occur at many steps Transcriptional control • Binding of RNA polymerase to promoter • Most critical step in regulation of most prokaryotic genes • Shift from initiation to elongation • Release of mRNA at termination Posttranscriptional control • Stability of mRNA • Efficiency of translation initiation • Stability of polypeptide 7th week General Genetics-BIO1053 Definitions • Constitutive – Synthesized all the time in fixed amounts – Does not respond to the environment • Adaptive – Responds to the environment • Inducible • Repressible • Induction: stimulation of protein synthesis • Inducer: molecule responsible for induction • Repression: inhibition of protein synthesis • Repressor: molecule responsible for repression Level of Arg biosynthetic enzymes Units β-gal + lactose No lactose Time Time Inducible – Produced in response to an inducer, usually the enzyme’s substrate 7th week + argenine Repressible – Repressed by the co-repressor, usually the endproduct General Genetics-BIO1053 Operon: Group of genes transcribed into a single mRNA Includes promoter & operator sequences The lactose operon in E coli The players • Three structural genes - lacZ, lacY, and lacA • Promoter - site to which RNA polymerase binds • Cis-acting operator site – controls transcription initiation • Trans-acting repressor - binds to the operator (encoded by lacI gene) • Inducer - prevents repressor from binding to operator 7th week General Genetics-BIO1053 Using lacZ to identify sets of genes regulated by the same stimulus Transposition of promoter-less lacZ coding region Library of clones containing lacZ insertions at random sites Screen library to identify all the genes that express lacZ in response to a signal Use of fusions to overproduce a gene product Expression of gene X under control of the lac regulatory system Expression of human growth hormone in E coli controlled by lac control region Regulation of the tryptophan (trp) operon in E coli Structural genes for tryptophan (Trp) biosynthesis are expressed only in the absence of Trp Two mechanisms for trp operon regulation • TrpR gene encodes the trp repressor that can bind to the Trp operator (TrpO) • When Trp is present, TrpR repressor binds to TrpO • When Trp is absent, TrpR repressor cannot bind to TrpO • Attenuation controls termination of transcription in the trp leader (TrpL) • When Trp is present, transcription terminates in TrpL • When Trp is absent, transcription doesn't terminate in TrpL Tryptophan acts as a corepressor Evidence that TrpR repressor is not the only regulator of the trp operon Constitutive expression of Trp biosynthesis doesn't occur in TrpR− mutants If TrpR were the sole regulator, maximal expression of trp genes would occur in the absence or presence of tryptophan Second regulatory mechanism is attenuation – control of gene expression by premature termination of transcription Transcription from the trp promoter produces two alternative mRNAs Attenuation controls termination of transcription in the trp leader (TrpL) • Truncated mRNA - terminates in TrpL, only 140 bases • Full-length mRNA - continues through TrpL and encodes all five structural genes Alternate stem-loop structures in trpL RNA Different regions of trpL have complementary base-pairing • Formation of the 1-2 stem-loop allows formation of the 3-4 stem-loop • Formation of the 2-3 stem-loop prevents formation of the 3-4 stem-loop • The 3-4 stem loop is a transcription terminator When tryptophan is present, transcription terminates The trpL mRNA is translated and includes two trp codons Movement of ribosomes through trpL mRNA depends on the availability of tRNATrp • When Trp is present, tRNATrp is available and rapid ribosome movement allows the formation of 3-4 stem-loop When tryptophan isn't present, transcription doesn't terminate in trpL Sigma factor (σ) recognition sequences Normal, housekeeping sigma factor is σ70 • Active under normal physiological conditions, but is inactivated by heat shock rpoH genes encodes σ32, an alternative sigma factor • Heat shock inducible genes have promoters that are recognized by σ32 • σ32 is resistant to inactivation by heat shock Sigma factor (σ) recognition sequences Binding of RNA polymerase to promoter The promoter sequence of the lac operon is TATGTT at the -10 region, and TTTACA at the -35 region The consensus sequence of σ70 which regulates the lac operon is TATAAT at the -10 region, and TTGACA at the -35 region They differ at three positions: two at the -10 region and one at the -35 region Experimentally, it has been found that binding of the RNA polymerase to the lac promoter is relatively weak Transcription of the lac operon often requires an activator such as catabolite activator protein (CAP) Translational control of another sigma factor encoded by the rpoS gene Under normal conditions, rpoS gene is transcribed but rpoS mRNA is not translated After stress response, a small RNA (dsrA) binds to rpoS mRNA and allows translation Medium contains glucose in limiting amounts and lactose 59 ... mutants clustered in the inducer-binding domain X-ray crystallography revealed the two separate domains 7th week General Genetics-BIO1053 Proteins act in trans, DNA sites act in cis Jacob and... most variable genes 7th week General Genetics-BIO1053 Chapter outline Overview of Prokaryotic Gene Regulation The Regulation of Gene Transcription Attenuation of Gene Expression: Termination of... superrepressor that binds to operator but cannot bind to the inducer In lacIS mutants, lac genes are repressed in the absence and the presence of inducer 7th week General Genetics-BIO1053 lacOc