Announcements 1. Today – in class problem solving: including handout/worksheet on recombinant DNA...

Announcements

1. Today – in class problem solving: including handout/worksheet on recombinant DNA technology, putting it all together AND restriction analysis problems

2. Based on your input, I’ve compiled slides from lectures that are good overviews of different topics covered. As time allows, I will go over “big picture” and main points of topics requested – in order of “popularity”.

3. Wednesday is review day – when I take your specific questions from problem sets, end of chapter problems, etc…

4. Exam 3 is Thursday, Friday, and Sunday at testing center. You will need a bluebook, pencil, calculator, ID – as usual.

5. GRE workshop Nov. 13 - Bovee UC 7pm- detail by main office

Restriction Mapping problems

**Note: fragments are linearized to start with.

Figure 18.23

Learning Check

What is the restriction map of this cloned DNA fragment, showing the locations of the restriction sites and relative distance between sites?

Linear DNA fragment

Marker EcoRI BamHI EcoRI/BamHI

15

9

6

10

8

7

5

8

6 5 4

2

Prokaryotic Promoter Lies Just Upstream (5’) of Transcribed Region; RNA Polymerase Binds Two Places

-10 TATA Box-35 Region

Anatomy of a Eukaryotic Gene (Protein Encoding)

TATA BoxCAAT Box

Cis-regulatory Elements may be located thousands of bases away; Regulatory TFs bind.

Pol II, Basal TFs bind

Transcriptionfactors

TBP-TATA binding proteinTAFs- TATA assoc. factors

RNA Processing in Eukaryotes

Pre-mRNA (primary transcript)

5’ cap and 3’ cleavage

Poly A tail added to 3 ‘end

Splicing: introns removed

Mature mRNA

Eukaryotic vs. Prokaryotic Transcription

• In eukaryotes, transcription and translation occur in separate compartments.

• In bacteria, mRNA is polycistronic; in eukaryotes, mRNA is usuallymonocistronic.

– Polycistronic: one mRNA codes for more than one polypeptide

– moncistronic: one mRNA codes for only one polypeptide

• 3 RNA polymerases in euk., 1 in prok. Binding of Basal Transcription Factors required for euk. RNA Pol II binding.

• Processing of mRNA in eukaryotes:

– 5’ 7-methylguanosine (7mG) cap added

– 3’ Poly-A tail added

– Splicing out of introns

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

Promoters: Eukaryotic vs. Prokaryotic

RNA pol II

RNA pol

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

Euk. - Chromatin and TFsProk. - Naked DNA and no TFs needed

Eukaryotic Enhancers and Promoters

Promoters- needed for basal level transcriptionEnhancers- 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

LiverRegulatoryTFs increasetranscriptionactivity

BrainNo 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.

Chromatin remodeling

TFs can recruit HATs or HDs

Bacterial Strategy

• If glucose is present,

– then use glucose as a carbon source.

• If glucose is not present, and if lactose is present,

– then use lactose (indirectly) as carbon source.

Levels of enzymes needed to use lactose as carbon source increase dramatically when lactose is present; enzymes are inducible and lactose is the inducer.

The Operon Model - components

R

L

pol

Repressor protein has 2 key binding sites

R

R

pol

pol

R R

pol

L

L

R

R

pol pol

pol pol

Learning Check

pol

R

L

Will transcription and translation of Z, Y,and A enzymes occur?

What would happen if a wild-type copy of I was added?

Catabolite Repression of lac Operon -/+ Glucose

Goal: efficiency, don’t waste energy converting lactose, when glucose available

Tryptophan Operon - Repressor Bindswhen tryptophan is present

Other Theoretical Possibilities

Separation of Nucleic Acids by CeCl Gradient Centrifugation

Meselson-Stahl Experiment

DNA Labeling with 15N

Subsequent Generations Labeled with 14N

Cesium Chloride Gradient Banding

Expected Results From Conservative or Dispersive Reproduction

If Conservative: Twobands, heavy and light,in 1st and 2nd generations

If Dispersive, one smearyband in 1st and 2ndgenerations

Expected Results if Semiconservative

These results were obtained.

A related experiment was performed in plants (Fig. 12.5)

Steps of DNA Synthesis

• Denaturation and Unwinding• Priming and Initiation• Continuous and Discontinuous Synthesis

– Including Proofreading and Error Correction

• Removal of Primer• Ligation of nicks in backbone

Continuous and Discontinuous Synthesis

• Continuouson Leading Strand. • Discontinuouson Lagging Strandcreates Okazakifragments.

• DNA ligase joinsnicks in backbone.

IV. The Eukaryotic Problem of Telomere Replication

RNA primer near end of the chromosome on lagging strand can’t be replaced with DNA since DNA polymerase must add to a primer sequence.

Do chromosomes get shorter with each replication???

Solution to Problem: Telomerase

• Telomerase enzyme adds TTGGGG repeats to end of lagging strand template.

• Forms hairpin turn primer with free 3’-OH end on lagging strand that polymerase can extend from; it is later removed.

• Age-dependent decline in telomere length in somatic cells, not in stem

cells (germ cells), cancer cells.

Aminoacyl tRNA synthetase

Charging the tRNA

Special Anticodon-Codon Base-Pairing Rules

Overview of Prokaryotic Translation

E P A

1

2

3