Biology 457 & 557 -  Week 5

 

Morphology of Eukaryotic Chromosomes

 

chromatin is further folded or organized into large units hundreds to thousands of kbs in length called

            condensation is the result of several orders of supercoiling

 

 

            nonhistone proteins are involved

 

why are nonhistones important?

 

 

 

these loops are anchored to this scaffold

 

 

a major protein of the scaffold has been identified as topoisomerase II

 

How do we know it is topo II?

 

 

 

binding sites for topo II are called

 

SARs were found to occur

 

evidence in Drosophila that chromatin uncoils in the transcribed region

 

 

 

So far we have talked about chromosomes as being linear structures composed of a single DNA molecule.  This DNA exists in a condensed or extended form and is associated with histones and scaffold proteins (nonhistones).  Although chromosomes differ in length and number among species, they all have to properly replicate and be stably inherited

 

 

 

In order to duplicate and segregate correctly, chromosomes must contain three functional elements (minimum requirements for a DNA molecule to function as a chromosome)

            1)

 

            2)

           

 

            3)

 

 

 

 

telomerase

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Spent time discussing the organization of genes, chromatin and chromosomes, now want

to discuss how genes are regulated

 

 

 

 

 

 

Molecular Basis of Transcriptional Regulation

 

discuss gene regulation/expression in prokaryotes and eukaryotes

 

the term gene expression commonly refers to the entire process

 

 

the most important control point in determining which genes are expressed and how much of the encoded mRNAs

 

 

there is an important difference between the purposes of gene regulation in single-celled versus multicellular organisms

            in bacteria

 

 

            most of the regulatory controls are easily reversible systems

 

 

            in eukaryotes

 

            precise decision so that the right gene is activated in the right cell at the right

            time

Transcriptional Regulation in Eukaryotes

you have studied transcription in general (DNA Ù RNA)

an organism’s DNA encodes all of the RNA and protein molecules

 

all RNA molecules are synthesized by RNA polymerase

            w prokaryotes

            w eukaryotes

eukaryotes differ from prokaryotes in 2 other important ways:

    1) eukaryotic RNA polymerase

    2) a single gene is controlled by several regulatory sequences

 

 

 

So how do the 100,000 or so genes in eukaryotic cells get turned on and off?

most cells retain

originally thought that cells differentiate because of

but now know that some type of regulation determines

 

regulation provides cells with a mechanism for determining

 

underlies the development and maintenance

            w differentiation

            w cells respond and adjust to stimuli

                        - heat shock proteins

                        - steroid hormones

 

Molecular basis of transcriptional is not completely understood

not simple, not controlled by 1 molecule

many regulatory proteins (also called nonhistones)

 

 

significant area of research

            w 100’s of regulatory proteins have been identified


 

Mention a few characteristics of regulatory proteins and how they control gene expression

1) to activate or repress transcription, these factors must be:

            w located in the nucleus

            w

           

2) regulatory proteins act by universal mechanisms

            w

           

 

3) eukaryotic regulatory factors are composed of modular components

            w

            DNA binding domain

           

            protein binding domain

 

            transcriptional activation domain

           

            however, not all regulatory proteins

 

4) almost all regulatory proteins are phosphorylated

            w protein kinases

            w also sensitive to where its added

 

5) other ways in which the activity of gene regulatory proteins are regulated

 

 

 

 

 

 

6) for genes encoding mRNAs, transcription factors and other regulatory proteins bind

    primarily to

 

            to better understand transcriptional regulation

 

 

 

 

 

 

important features or elements:

a)      multimeric complex

 

            b)     TATA box in promoter

           

            c)     CAAT sequences in enhancers (regulatory sequences) upstream or

 

 

            d) CG boxes

 

            e) enhancers and promoters

                        - action-at-a-distance phenomenon

                        - enhancer-bound regulatory proteins can interact with general

                           transcription factors

 

 

OVERALL, NO ELEMENT IS COMMON TO ALL EUKARYOTIC GENE CONTROL REGIONS, BUT REGULATION PROBABLY REQUIRES 1 OR MORE OF THESE ELEMENTS

 

 

another simple example to show how these elements interact

            GAL4/GAL80 regulatory proteins of yeast that regulate galactose metabolism

 

 

 

 

 

 

 

 

 

 

 

a) binding of GAL4 to enhancer

b) interaction of enhancer and promoter

c) transcription starts

d) the repressor protein GAL80 binds to GAL4

e) when galactose present, GAL80 undergoes conformational change

 

7) gene expression is controlled by the cooperative regulation of multiple proteins

 

 

 

 

 

 

coordinated control of serum albumin gene (protein of blood plasma) by multiple proteins

 

 

 

 

 

 

 

 

in most eukaryotes, gene control requires both widely distributed factors and

    factors that have a limited, cell-specific distribution

 

8) many eukaryotic genes are controlled in networks

           

                        a) steroid hormone receptors - these regulatory proteins (found in the nucleus) bind steroid hormones    (estrogen, progesterone, testosterone)

 

                        response elements

 

 

                        b) homeotic genes - control major developmental, differentiation, and

                           segmentation pathways

                                    - discovered first in Drosophila

                                   

                                    - encode for regulatory proteins that recognize an 8 bp sequence

                                      in the 5¢-flanking region of genes

        homeodomain

        octamer sequence

                        c) heat shock genes - activated by adverse conditions of stress

                                   

                                            heat shock factors

                                   

                                      called heat shock elements

 

presented examples which outline the activation of specific genes but gene regulation

     is much more complex

 

What about mechanisms that modify transcription in general, rather than particular genes

DNA does not exist as a linear molecule with no obstruction to transcription

 

 

DNA is packaged in a nucleus

 

 

ultimately it is this packaging of DNA by proteins and associated modifications that

     play a major role in gene regulation

 

Chromatin structure and the control of gene expression

 

 

 

 

 

 

 

 

can you see a problem presented by folding and activation of transcription?

 certain mechanisms must be used to place a gene or genes in a transcriptionally-ready

 

 conversely, modifications to DNA and associated proteins (histones) can repress