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Announcements. Tuesday afternoon lab section: lab start time next week is 3pm. 2-3 pm might be a good time to do problem set 6! 2. No advance reading for next week’s lab; focus on your lab report. 3. Problem set 5 due at start of class today. 4. Reading - Ch. 16: skip btm. 442- top 444.

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Announcements

Tuesday afternoon lab section: lab start time

next week is 3pm. 2-3 pm might be a good time to do problem set 6!

2. No advance reading for next week’s lab; focus on your lab report.

3. Problem set 5 due at start of class today.

4. Reading - Ch. 16: skip btm. 442- top 444.


Review of Last Lecture

1.The lac operon - in detail; know roles of all components

  • lactose

  • repressor protein from I gene

  • Operator sequence

  • Promoter sequence

  • Regulation of expression of 3 structural genes:

  • lacY, lacZ, lacA

  • CAP + cAMP

  • Glucose

  • **Clarification: maximal transcription= repressor bound by lactose and CAP bound to CAP-binding site

    2. The trp operon - very briefly


Learning Check

  • Will B-galactosidase be made: a-always, b-never, c-sometimes (in presence of lactose)

    1. I+O+Z+

    2. I+OCZ+

    3. ISO+Z+


Outline of Lecture 27

I. Eukaryotic Gene Expression: it’s more complicated being multicellular

II. The Promoter

III. Enhancers

IV. Methylation

V. Alternative splicing


I. Problems of Multicellularity

  • All of our genes are present in every cell, but only certain proteins are needed.

  • Expression of a gene at the wrong time, in the wrong type of cell, or in abnormal amounts can lead to deleterious phenotypes or death - even when the gene itself is normal.

Pancreatic cell Neuron

+ insulin - insulin

- neurotransmitter +neurotransmitter


Levels of Regulation of Eukaryotic Gene Expression

  • “What is true of E. coli is only partly true of elephants.”

  • Prokaryotic control primarily at the transcription level.

  • Eukaryotic control at several levels


Levels of

gene

regulation

***focus today


The interphase nucleus

Chromosome structure is continuously rearranged, so that transcriptionally active genes are cycled to edge of territories.


Organization/ packaging of DNA

Nucleus= 5-10 m

(0.01mm)

Diploid genome= 6.4x109 bp

0.34nm/bp

DNA=2 meters

(2000 mm)



II. Promoters: Eukaryotic vs. Prokaryotic

RNA pol II

RNA pol

Promoters: sequences adjacent to genes, where RNA pol binds to initiate transcription

Euk. - Chromatin and TFs

Prok. - Naked DNA and no TFs needed



Transcription Regions of DNA for Gene Expression

factors

TBP-TATA binding protein

TAFs- TATA assoc. factors


III. Eukaryotic Enhancers and Promoters Regions of DNA for Gene Expression

Promoters- needed for basal level transcription

Enhancers- needed for full level transcription; location and orientation variable

Two types of transcription factors bind enhancers and affect levels of txn: true activators and anti-repressors


Combinatorial Model of Gene Expression Regions of DNA for Gene Expression

Liver

Regulatory

TFs increase

transcription

activity

Brain

No reg.TFs in this cell for albumin enhancer


Binding of True Activator TFs to Enhancers Greatly Stimulates Transcription

Looping of DNA allows Activator TF bound to Enhancer to interact with Promoter, facilitating binding of Basal TF complex.


Types of Regulatory Transcription Factors Stimulates Transcription

  • True activators are modular proteins: one domain binds DNA in enhancer; one domain interacts with protein at promoter

  • Classified by DNA-binding motifs in the protein:

    • Helix-Turn-Helix

    • Zinc Finger

    • Leucine Zipper

    • Helix-Loop-Helix


Helix-Turn-Helix (HTH) TFs Stimulates Transcription

  • Two -helical stretches of AA’s linked by non-helical sequence of AA’s

  • e.g. lac repressor protein

    (binds operator DNA)

  • also eukaryotic homeodomain TFs, important in embryonic development


Zinc Finger TFs Stimulates Transcription

  • Zn2+ is coordinated between His and Cys residues in protein, forming “finger” of protein

  • 2-13 fingers may be present

  • Found in pseudooncogenes and in DrosophilaKruppel TF in embryonic development


Leucine Zipper TFs Stimulates Transcription

  • Dimeric, held together by interactions between leucine residues

  • includes AP-1 TF, a heterodimer of Jun and Fos proteins, important in control of cell division; mutation can cause cancer.


Helix-Loop-Helix (HLH) TFs Stimulates Transcription

  • Dimeric: two subunits of two helices linked by loops

  • includes mammalian TFs for muscle differentiation - MyoD, myogenin and Myf-5


Antirepressor Transcription Factors Stimulates Transcription

Access

Slow txn


TFs can recruit HATs or HDs Stimulates Transcription


IV. Control of gene expression Stimulates Transcriptionby DNA Methylation

  • Addition of CH3 to selected C’s in DNA can inactivate genes, e.g. high levels are seen in inactivated X chromosome of female mammals.

  • Mammals have about 5% methylation.

  • Not essential in eukarotyes, since Drosophila has 0% methylation.

  • First observed in lac operon: methylation of operator DNA sequence affects binding by repressor


V. Post transcriptional gene regulation Stimulates Transcription

If humans have approximately the same number of genes as a fruit fly, and we require more complex cellular functions (presumably with a larger number of proteins) - how do we accomplish this?


Alternative splicing Stimulates Transcription

1. Chromosomal ratio activates txn of Sxl in females only

2. SXL controls splicing of tra-2 mRNA

3. Females: exon 2 (which has a stop codon) is removed via SXL

Males: exon 2 is not removed.

Males: no active TRA

Females: TRA is made.

5. TRA directs splicing of dsx mRNA in specific manner; in males default splicing occurs.


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