GENE REGULATION OXYTOCIN The following gene is from

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GENE REGULATION

GENE REGULATION

OXYTOCIN • The following gene is from m. RNA that has been copied with

OXYTOCIN • The following gene is from m. RNA that has been copied with reverse transcriptase to produce c. DNA • Sequence provided is from the coding strand

1 accagtcacg gaccctggac ccagcgcaccatgg ccggccccag cctcgcttgc 61 tgtctgctcg gcctcctggc gctgacctcc gcctgctaca tccagaactg ccccctggga 121

1 accagtcacg gaccctggac ccagcgcaccatgg ccggccccag cctcgcttgc 61 tgtctgctcg gcctcctggc gctgacctcc gcctgctaca tccagaactg ccccctggga 121 ggcaagaggg ccgcgccgga cctcgacgtg cgcaagtgcc tcccctgcgg ccccgggggc 181 aaaggccgct gcttcgggcc caatatctgc tgcgcggaag agctgggctg cttcgtgggc 241 accgccgaag cgctg ccaggaggag aactacctgc cgtcgccctg ccagtccggc • 301 cagaaggcgt gcgggagcgg gggccgctgc gcggtcttgg gcctctgctg cagcccggac • 361 ggctgccacg ccgaccctgcgacgcg gaagccacct tctcccagcg ctgaaacttg • 421 atggctccga acaccctcga agcgcgccac tcgcttcccc catagccacc ccagaaatgg • 481 tgaaaataaagcagg tttttctcct ct

SIGNAL SEQUENCE • 37 -93 • Before signal sequence is the leader sequence •

SIGNAL SEQUENCE • 37 -93 • Before signal sequence is the leader sequence • Mature Oxytocin 94 -120 27 nucleotides = 9 amino acids

 • Preproprotein 37 - 411 • Signal seq, oxytocin + neurophysin I Proprotein

• Preproprotein 37 - 411 • Signal seq, oxytocin + neurophysin I Proprotein 94 -411 Oxytocin + neurophysin I • Mature Peptides modified from Proprotein • Trailer sequence responsible for Poly. A-polymerase action

1 accagtcacg gaccctggac ccagcgcaccatgg ccggccccag cctcgcttgc 61 tgtctgctcg gcctcctggc gctgacctcc gcctgctaca tccagaactg ccccctggga 121

1 accagtcacg gaccctggac ccagcgcaccatgg ccggccccag cctcgcttgc 61 tgtctgctcg gcctcctggc gctgacctcc gcctgctaca tccagaactg ccccctggga 121 ggcaagaggg ccgcgccgga cctcgacgtg cgcaagtgcc tcccctgcgg ccccgggggc 181 aaaggccgct gcttcgggcc caatatctgc tgcgcggaag agctgggctg cttcgtgggc 241 accgccgaag cgctg ccaggaggag aactacctgc cgtcgccctg ccagtccggc • 301 cagaaggcgt gcgggagcgg gggccgctgc gcggtcttgg gcctctgctg cagcccggac • 361 ggctgccacg ccgaccctgcgacgcg gaagccacct tctcccagcg ctgaaacttg • 421 atggctccga acaccctcga agcgcgccac tcgcttcccc catagccacc ccagaaatgg • 481 tgaaaataaagcagg tttttctcct ct

Notice the 1 st 3 nucleotides… • CODING STRAND ATG TEMPLATE STRAND TAC m.

Notice the 1 st 3 nucleotides… • CODING STRAND ATG TEMPLATE STRAND TAC m. RNA AUG…start codon Amino Acid Methionine (Met)

tgc tac atc cag aac tgc ccc ctg gga acg atg tag gtc ttg

tgc tac atc cag aac tgc ccc ctg gga acg atg tag gtc ttg acg ggg gac cct ugc uac auc cag aac ugc ccc cug gga Cys-tyr-ile-gln-asn-cys-pro-leu-gly

WOBBLE EFFECT • rd 3 base of t. RNA may form H -bonds with

WOBBLE EFFECT • rd 3 base of t. RNA may form H -bonds with more than 1 kind of nucleotide • Ie AAU and AAC Asn

TRANSCRIPTION FACTORS

TRANSCRIPTION FACTORS

RECOGNIZE SEQUENCES • Transcription factors recognize DNA sequences inorder to target specific genes

RECOGNIZE SEQUENCES • Transcription factors recognize DNA sequences inorder to target specific genes

ROLE OF REGULATORY PROTEINS • Transcription factors are genetic switches • Master Genes ie

ROLE OF REGULATORY PROTEINS • Transcription factors are genetic switches • Master Genes ie HOX genes code for transcription factors • Related “Regulatory Proteins” in prokaryotes

PROKARYOTIC REGULATION • No hormonal regulation • No introns no splicing • No capping/tailing

PROKARYOTIC REGULATION • No hormonal regulation • No introns no splicing • No capping/tailing • Coupled transcription/translation

 • Constitutive – always on; can be regulated (enzymes in glycolysis) • Inducible

• Constitutive – always on; can be regulated (enzymes in glycolysis) • Inducible – off but can be switched on • Repressible – on but can be switched off

OPERONS • Cluster of genes in which expression is regulated by operatorrepressor protein interactions,

OPERONS • Cluster of genes in which expression is regulated by operatorrepressor protein interactions, operator region, and the promoter. • Promoter • Repressor • Operator (controlling site) • Coding sequences • Terminator

Lac Operon • Lactose is an inducer • “Inducible” operon • Negative regulation

Lac Operon • Lactose is an inducer • “Inducible” operon • Negative regulation

– -galactosidase (lac. Z) • Breaks lactose into glucose + galactose. • Converts lactose

– -galactosidase (lac. Z) • Breaks lactose into glucose + galactose. • Converts lactose to the allolactose, regulates lac operon. –Lactose permease (lac. Y) • Transports lactose across cytoplasmic membrane. –Transacetylase (lac. A)

 • Positive control when lactose is E. coli’s sole carbon source (but not

• Positive control when lactose is E. coli’s sole carbon source (but not if glucose also is present). • Catabolite activator protein (CAP) binds c. AMP, activates, and binds to a CAP recognition site upstream of the promoter (c. AMP is greatly reduced in presence of glucose).

 • CAP changes the conformation of DNA and facilitates binding of RNA polymerase

• CAP changes the conformation of DNA and facilitates binding of RNA polymerase and transcription. • When glucose and lactose are present, E. coli preferentially uses glucose due to low levels of active CAP (low c. AMP).

 • Regulation of the trp operon: • “repressible” gene • 1. Repressor/operator interaction

• Regulation of the trp operon: • “repressible” gene • 1. Repressor/operator interaction • When tryptophan is present, tryptophan binds to trp. R gene product. • trp. R protein binds to the trp operator and prevents transcription.

 • Is TRP operon neg or pos regulation? • What type of regulation

• Is TRP operon neg or pos regulation? • What type of regulation controls repressors/inducers?

Jacob and Monod • What is true for E. coli is also true for

Jacob and Monod • What is true for E. coli is also true for the elephant!

GENETIC SWITCHES • A switch includes. . a) Binding protein b) Binding protein recognizes

GENETIC SWITCHES • A switch includes. . a) Binding protein b) Binding protein recognizes a strecth of DNA

Significance? • Mutate the gene encoding the transcription factor or the DNA sequence to

Significance? • Mutate the gene encoding the transcription factor or the DNA sequence to which it binds and gene expression can be altered!

GENE INACTIVATION • 1. CHROMATIN structure – Heterochromatin (tight– gene off) vs Euchromatin (loose

GENE INACTIVATION • 1. CHROMATIN structure – Heterochromatin (tight– gene off) vs Euchromatin (loose – gene on) • 2. Methylation – adding methyl group to inactivate genes on DNA • 3. small RNA affects chromatin structure / interferes with transcription (RNAi system)

Methylation • Reinforces inactivation • Involved in Barr-Body (inactive X chromosome)

Methylation • Reinforces inactivation • Involved in Barr-Body (inactive X chromosome)

TRANSCRIPTIONAL CONTROL • Signals (hormones) in eukaryotes • Environmental- heat shock proteins • Regulator

TRANSCRIPTIONAL CONTROL • Signals (hormones) in eukaryotes • Environmental- heat shock proteins • Regulator proteins (Transcription factors) – bind to TATA (like prokaryotes) • UPEs (Upstream Promoter Elements) – increase efficiency of RNA pol.

Example… • Glucocorticoids released by stress bind to steroid receptor (in liver) forms complex

Example… • Glucocorticoids released by stress bind to steroid receptor (in liver) forms complex binds to DNA activates genes involved in gluconeogenesis

Weakly transcribed

Weakly transcribed

Strongly transcribed

Strongly transcribed

 • TRANSLATIONAL CONTROL • CAPPING – 7’ methyl guanosine added to 5’ role

• TRANSLATIONAL CONTROL • CAPPING – 7’ methyl guanosine added to 5’ role in initiation/ protection • Poly A tail protection, promotes transport out of Nucleus • Splicing – Introns removed Exons for code

 • Differential m. RNA processing –Cells in each tissue produce own version of

• Differential m. RNA processing –Cells in each tissue produce own version of m. RNA –For example, different forms of troponin, a protein that regulates muscle contraction, produced in different muscle tissue

 • POST TRANSLATIONAL • modification of polypeptide (ie reduction in size of proinsulin

• POST TRANSLATIONAL • modification of polypeptide (ie reduction in size of proinsulin to insulin) • chemical modification – addition of phosphate (Kinases) removal of phosphate (Phosphatases)

 • Examine the insulin pathway as a form of gene regulation

• Examine the insulin pathway as a form of gene regulation