AP Biology 2007-2008 Control of Eukaryotic Genes – Chapter 19

AP Biology 2007-2008

Control of Eukaryotic Genes –Chapter 19

The BIG Questions… How are genes turned on & off

in eukaryotes? How do cells with the same genes

differentiate to perform completely different, specialized functions?

Evolution of gene regulation Prokaryotes

single-celled evolved to grow & divide rapidly must respond quickly to changes in

external environment exploit transient resources

Gene regulation turn genes on & off rapidly

flexibility & reversibility adjust levels of enzymes

for synthesis & digestion

Evolution of gene regulation Eukaryotes

multicellular evolved to maintain constant internal

conditions while facing changing external conditions

homeostasis regulate body as a whole

growth & development long term processes

specialization turn on & off large number of genes

must coordinate the body as a whole rather than serve the needs of individual cells

Points of control The control of gene

expression can occur at any step in the pathway from gene to functional protein1. packing/unpacking DNA

2. transcription

3. mRNA processing

4. mRNA transport

5. translation

6. protein processing

7. protein degradation

How do you fit all that DNA into nucleus?

DNA coiling & folding

double helix nucleosomes chromatin fiber looped

domains chromosome

from DNA double helix to condensed chromosome

1. DNA packing

Nucleosomes “Beads on a string”

1st level of DNA packing histone proteins

8 protein molecules positively charged amino acids bind tightly to negatively charged DNA

DNA packing movie

8 histone molecules

DNA packing as gene control Degree of packing of DNA regulates

transcription tightly wrapped around histones

no transcription genes turned off

heterochromatindarker DNA (H) = tightly packed

euchromatinlighter DNA (E) = loosely packed

H E

DNA methylation Methylation of DNA blocks transcription factors

no transcription genes turned off

attachment of methyl groups (–CH3) to cytosine C = cytosine

nearly permanent inactivation of genes ex. inactivated mammalian X chromosome = Barr body

Histone acetylation Acetylation of histones unwinds DNA

loosely wrapped around histones enables transcription genes turned on

attachment of acetyl groups (–COCH3) to histones conformational change in histone proteins transcription factors have easier access to genes

2. Transcription initiation Control regions on DNA

promoter nearby control sequence on DNA binding of RNA polymerase & transcription

factors “base” rate of transcription

enhancer distant control

sequences on DNA binding of activator

proteins “enhanced” rate (high level)

of transcription

Model for Enhancer action

Enhancer DNA sequences distant control sequences

Activator proteins bind to enhancer sequence &

stimulates transcription Silencer proteins

bind to enhancer sequence & block gene transcription

Turning on Gene movie

Transcription complex

Enhancer

ActivatorActivator

Activator

Coactivator

RNA polymerase II

A

B F E

HTFIID

Core promoterand initiation complex

Activator Proteins• regulatory proteins bind to DNA at distant

enhancer sites• increase the rate of transcription

Coding region

T A T A

Enhancer Sitesregulatory sites on DNA distant from gene

Initiation Complex at Promoter Site binding site of RNA polymerase

3. Post-transcriptional control Alternative RNA splicing

variable processing of exons creates a family of proteins

4. Regulation of mRNA degradation Life span of mRNA determines

amount of protein synthesis mRNA can last from hours to weeks

RNA processing movie

RNA interference Small interfering RNAs (siRNA)

short segments of RNA (21-28 bases) bind to mRNA create sections of double-stranded mRNA “death” tag for mRNA

triggers degradation of mRNA cause gene “silencing”

post-transcriptional control turns off gene = no protein produced

NEW!

siRNA

Action of siRNA

siRNA

double-stranded miRNA + siRNA

mRNA degradedfunctionally turns gene off

Hot…Hotnew topicin biology

mRNA for translation

breakdownenzyme(RISC)

dicerenzyme

QuickTime™ and aTIFF (Uncompressed) decompressor

are needed to see this picture.

RNA interference1990s | 2006









Andrew FireStanford

Craig MelloU Mass



“for their discovery of RNA interference —gene silencing by double-stranded RNA”

5. Control of translation Block initiation of translation stage

regulatory proteins attach to 5' end of mRNA

prevent attachment of ribosomal subunits & initiator tRNA

block translation of mRNA to protein

Control of translation movie

6-7. Protein processing & degradation Protein processing

folding, cleaving, adding sugar groups, targeting for transport

Protein degradation ubiquitin tagging proteasome degradation

Protein processing movie

Ubiquitin “Death tag”

mark unwanted proteins with a label 76 amino acid polypeptide, ubiquitin labeled proteins are broken down

rapidly in "waste disposers" proteasomes

1980s | 2004

Aaron CiechanoverIsrael

Avram HershkoIsrael

Irwin RoseUC Riverside

Proteasome Protein-degrading “machine”

cell’s waste disposer breaks down any proteins

into 7-9 amino acid fragments cellular recycling

play Nobel animation

initiation of transcription

1

mRNA splicing

2

mRNA protection3

initiation of translation

6

mRNAprocessing

5

1 & 2. transcription - DNA packing - transcription factors

3 & 4. post-transcription - mRNA processing

- splicing- 5’ cap & poly-A tail- breakdown by siRNA

5. translation - block start of translation

6 & 7. post-translation - protein processing - protein degradation

7 protein processing & degradation

4

4

Gene Regulation

DNA TechnologyChapter 20

Quick Definitions:

Genetic engineering – the direct manipulation of genes for practical purposes.

Biotechnology – manipulation of organisms or their components to perform practical tasks or provide useful products.

We will now look at many different techniques that assist us in these two fields:

GOAL: To make recombinant DNA and put it to use.

Producing Recombinant DNA

Gene cloning – getting well-defined, gene-sized pieces of DNA and making multiple copies that we can put to use. This is a multi-step process.

Restriction enzymes – Discovered in bacteria in 1960’s They cut foreign DNA at a restriction site

(specific recognition sequence). Produces restriction fragments.

Restriction EnzymesRestriction Site

Sticky Ends

How do we insert our gene?

Cloning vectors – DNA molecules that carry foreign DNA into a cell and replicate. i.e. viruses, plasmids

How do you tell if it is successful?

Grow plates on antibiotic impregnated plates

Nucleic acid hybridization – Figure 20.4

Figure 20.3

Nucleic Acid Hybridization

1. Transfer cells to filter.

2. Treat cells to denature DNA on filter (chemical or heat)

3. Add radioactive / fluorescent probe to filter.

4. Take a picture (autoradiography).

5. Return to original culture and select those colonies that showed radioactivity / fluorescence.

Genomic Libraries

When a gene is cloned, not just the desired gene is inserted into the bacteria cells. There may be many other genes of interest, so you can save these bacteria with the recombinant genes for later experiments in a genomic library.

cDNA library – this is made from the mRNA present = the genes being transcribed. You produce the complimentary DNA from the mRNA so you only get the coding sequences.

PCR

Polymerase Chain Reaction

Analysis of cloned DNA

NOW WHAT? We have all this DNA, how do we make sense out of it?

Gel electrophoresis

Southern Blot – More specific E. M. Southern – 1975 Steps:

1. Take DNA from source, splice.

2. Load fragments into gel and separate by electrophoresis.

3. Blotting – takes DNA sample from gel and transfers to paper.

4. Hybridize – use radioactive probe and develop film.

5. Study bands and the differences that may exist.

More techniques…

RFLP’s – restriction fragment length polymorphisms – differences that exist in the non-coding sequences of genes.

In-Situ Hybridization – allows you to locate specific gene on a chromosome in a genome. Much like labeling your DNA with a probe Southern blotting.

Mapping Entire Genomes

Baker’s yeast6,000

Fruit fly13,000

Roundworm19,000

Mustard25,000

Human35,000

Human Genome Project

Officially began in 1990 – international effort to map the entire human genome, determining every nucleotide sequence.

Types of Mapping: Genetic (linkage) Mapping – mapping the genome

with genetic markers as reference points Physical Mapping – determine the distance

between markers – cut DNA into manageable fragments

DNA Sequencing – determine the sequence of these fragments.

What did we learn?

Only about 3% of our DNA is protein-coding. The number of human genes is far smaller than

previously thought The absolute number of human proteins remains

unknown but far exceeds the number of genes. Very few genes (~1%) are “uniquely human” – almost

all are seen in other organisms. On average, the DNA sequence of one person differs

from another once every 700 nucleotides.

Applications of DNA Technology

Medicine: Diagnosis of

disease Human gene

therapy Pharmaceutical

products

Applications of DNA Technology

Forensics DNA Fingerprinting

Agricultural Transgenic

organisms Transgenic plants

Environmental Bioremediation Nitrogen fixation

“power houses”

Documents

AP Biology 2007-2008 Control of Eukaryotic Genes – Chapter 19