Molecular Biology

• Largely Concerned with Gene Expression

• What Turns it On/Off?

• How that is Achieved?

• How Much?

Regulation of Gene Expression - 2 lectures

In Eukaryotes Regulation of Gene Expression is Complex - Not just on and off -

Vary magnitude

Regulation of Gene Expression

1 - Revision of Eukaryotic Gene Structure

2- Need for Global regulation of genes

Molecular cell Biology 5th Edition

Mitochondria structure, aerobic and anaerobic metabolismpp 171-172, 304-312

Gene structure pp 405 -408

Molecular Genetic Techniques and CloningChapter 9

Journal of Experimental BiologyVolume 201, pp 1177-1195 (1998)Kwast et al.

Regulation of Geneexpression in Eukaryotes

Gene regulation in prokaryotes and/orsingle cellular organisms is different inthat gene regulation is primarilyconcerned with responding to externalstimuli. These include nutrients,temperature.

Although multicellular organsims alsorespond to such stimuli, eg plants,another level of gene regulation isinvolved.

For multicellular organisms the rightgene must be activated at the right time inthe right cell.

Examples of gene regulation at all levelshave been documented, transcriptional

Eukaryotic messages have 3’ untranslatedregion

can also vary in size from tens tohundreds of bases

Also have a poly A tail

A series of A residues that are added in a nontemplate dependant manner aftertranscription

The amount of information in EukaryoticDNA is 1 X 109 bp

Estimated to be 35, 000 genes

The function of non-coding DNA is notknown, obviously some of the non-codingDNA acts as “promoter” regions etc.However still lots of DNA that appears tohave no apparent function.

Gene - Is a piece of DNA that encodes a functional unit

- remember have ribosomal and tRNA genes so genes not restricted to encoding proteins

Central Dogma of molecular biology is thatDNA is the genetic material that is replicatedand passed onto succeeding generations. It istranscribed into RNA, which is subsequentlytranslated into protein.

RNA - Three types

Ribosomal RNA (rRNA) - as namesuggests found in ribosomes whichfunction to synthesise proteins

Messenger RNA (mRNA) - This type ofRNA specifies the sequence of amino acidsin a protein by triplet codon bases. ThemRNA sequence is translated into aprotein sequence.

Transfer RNA (tRNA) - This RNA acts asan intermediate between the mRNA andprotein. Through complementary basepairing to the mRNA it delivers the aminoacid coded in the mRNA to the ribosome.

Proteins - all proteins are encoded for bymRNA and synthesised on ribosomes. Thesehave various functions including structural(collagen, cell membrane proteins), enzymatic(digestive, intracellular metabolism), signals(insulin) and defence (antibodies).

Obviously get exception to this flow ofinformation1) RNA viruses2) RNA editing3) Ribozymes4) Prions

DNA

RNA

Protein

Transcription

Translation

Replication

DNA

RNA

Protein

Transcription

Translation

Reverse

Transcription

If RNA is reversed transcribed to produceDNA, the DNA is called cDNA, as it iscomplementary to the RNA copy.

No process of reverse translation known todate

N C

Protein

mRNA

Genomic DNA

5 3

5 3

Relationship between the structure of protein,mRNA and Genes

This is not the structural relationshipbetween Protein, mRNA and genomic DNA inEukaryotic cells

The relationship is far more complicated andit is important to get the terminology correctas you will come across it in texts etc.

N C

Protein

mRNA

5 3

AUG UAA

Start Stop

AAAA

Eukaryotic messages have a 5’ Cap

Also have a 5 ‘ untranslated regioncan Vary from tens to hundreds of bases

mRNA

5 3

AUG UAA

Start Stop

AAAA

Genomic DNA

Again this is not the relationship betweenmRNA and genomic DNA

mRNA

5 3

AUG UAA

Start Stop

AAAA

Exon 1 Exon 2 Exon 3 Exon 4Intron 1 Intron 2 Intron 3

Genomic genes in Eukaryotes containintervening sequences known as Introns.

Therefore the initial RNA transcript containsboth exons and introns, the introns areremoved from the primary RNA transcript ina process call splicing

How do we know where translation starts inthe mRNA

First in frame AUG

Not necessarily the first AUG but the first inframe AUG

Find this from:I) Protein sequence - care as lots ofeukaryotic proteins are processed

ii) Sequence analysis on a computer

How do we know where transcriptionalbegins -

Transcriptional start site

Need to know before we can start aboutpromoter elements etc

Two methods1) S1 nuclease mapping2) Primer extension

Will use control of gene expression by oxygen as an example

• Take place in all organisms - fungi, plants and animals

• Critical for survival

• Evolved early so can use comparative approachesto Understand

Oxygen is Toxic

QuickTime™ and aTIFF (LZW) decompressor

are needed to see this picture.

Will Not deal with Reactive Oxygen Species - Rather will deal withRole of oxygen in respiration - Oxidative Phosphorylation

Aerobic respiration essential for survival of multi-cellular Organisms

Some micro-organisms can survive anaerobically

Anaerobic - yeast - Industrial applications

Plants - Loss of Yield - results in large loss of life and/or commercial income

Animals - Medical conditions - hear disease, cancer etc

Genome Information

S. cerevisiae C. elegans A. thaliana H. sapiens

# Cells 1 ~1000 >1x106 >1x106

Size 12Mbp 97Mbp 125Mbp 3.2 Gbp

Chromosomes 16 6 5 23

Predicted ORFs ~6,000 ~19,000 ~28,000 ~35,000

%Coding 72% 27% 50% 1.5%

Aerobic metabolism:(oxidative metabolism)

Glycolysis TCA cycle oxidative phosphorylation

Anaerobic metabolism:(fermentation)

Glucose Acetyl CoA Ethanol

Diauxic shift:

Metabolic change as fermentable carbon source is used up from…

Glucose Fermentative (Glycolysis Ethanol)

Oxidative Metabolism(Ethanol TCA cycle)

to…

yeast carbon metabolism

• Two membranes

• Inner membrane invaginated

• Numbers of mitochondria per cell vary but usually 100s/cell

Matrix contains the TCA cycle (and other) soluble enzymes

Inner membrane contains metabolite transporters and the electron transport chain

Mitochondrial structure

Overview of aerobic

respiration

Four large, multi-subunit protein complexes

- complex I is a NADH-ubiquinone reductase- complex II is succinate dehydrogenase (part of the TCA cycle)- complex III is the ubiquinone -cytochrome c reductase- complex IV is cytochrome oxidase

The respiratory electron transport chain

NADH + CO2

One pyruvate molecule is completely oxidised to CO2

4-Carbons

3-Carbons

CO2

6-Carbons

NADH + CO2

NADH

FADH

Outline of Tricarboxylic Acid Cycle

The NADH and FADH produced are oxidised by the respiratory electron transport chain

Mitochondria have their own DNA and RibosomesMitochondria have some of their own DNA, ribosomes, and can make many of their own proteins. The DNA is circular and lies in the matrix in structures called "nucleoids".  Each nucleoid may contain 4-5 copies of the mitochondrial DNA (mtDNA).

mitochondrialDNA

Human mtDNA

• small, double stranded circular chromosome

• 16,569 bp in total

• no non-coding DNA

• no introns

• polycistronic replication which is initiated from the D (displacement)- loop region

• followed by splicing of transcript to form messages.

Organisation of the mitochondrial chromosome

human mtDNA

yeast mtDNA

Yeastmitochondrial chromosome

maize mitochondrial genome

In all organisms, only a few of the proteins of the mitochondrion are encoded by mtDNA, but the precise number varies between organisms

• Subunits 1, 2, and 3 of cytochrome oxidase• Subunits 6, 8, 9 of the Fo ATPase• Apocytochrome b subunit of complexIII• Seven NADH-CoQ reductase subunits (except in yeast)

The nucleus encodes the remaining proteins which are made in the cytosol and imported into the mitochondrion.

Most of the lipid is imported.

Synthesis of mitochondrial proteins

c

UQUQ

1/2

O 2

H 2 O

II

III

IV

V

F 1

F o

NADH

NAD+

H+

H+

H+H+

ADP + Pi

ATP

= Nuclear Encoded

= Mitochondrial Encoded

succinate

fumarate

I II

Proteins typically encoded in mitochondrion

Mitochondria are largely maternally inherited in higher Mitochondria are largely maternally inherited in higher animals and plantsanimals and plants

In mammals, most of the mitochondrial DNA (mtDNA) is inherited from the mother.  This is because the sperm carries most of its mitochondria its tail and has only about 100 mitochondria compared to 100,000 in the oocyte.  

Although sperm mitochondria penetrate the egg, most are degraded after a few hours. As the cells develop, more and more of the mtDNA from males is diluted out.  Hence less than one part in 104 or 0.01% of the mtDNA is paternal.

Mitochondria are largely maternally inherited in Mitochondria are largely maternally inherited in higher animals and plantshigher animals and plants

This means that mutations of mtDNA are passed from mother to child.  It also has implications for the cloning of mammals with the use of  somatic cells.  The nuclear DNA would be from the donor cell, but the mtDNA would be from the host cell.  This is how Dolly the sheep was cloned.  

In plants, the cytoplasm, including the mitochondria and the plastids, are contributed only by the female gamete and not by the pollen - again, mutations in organelle DNA are inherited maternally.

• Mitochondria divide by fission and are not made de novo

Human Evolution and mtDNAHuman Evolution and mtDNA

• they are inherited mainly from the mother: >99% of our mitochondria are derived from those (1000 or so) present in our mother’s ovum

D-loop: origin of mtDNA replication

Human evolution can be traced by analysis of the base sequence in a small part of the mitochondrial genome which does not encode a gene and which is quite variable.

- the so-called D-loop.

Human Evolution and mtDNAHuman Evolution and mtDNA

The D-Loop of the mtDNA is the start of replication/transcription site and contains 400-800 bp

Unlike the rest of mtDNA in humans, which is highly conserved, this region is very variable between people

It also has a very high frequency of change during evolution (about 2% per million years)

Human Evolution and mtDNAHuman Evolution and mtDNA

By comparing different groups, we can get a glimpse of human evolutionary lines.

Eg, African individuals have more variability between each other than do Asians, indicating that the former have had more time to accumulate changes - ie, the Africans are a more ancient group.

Human Evolution and mtDNAHuman Evolution and mtDNA

This makes the D-loop a very powerful tool for the study of evolutionary relationships between organisms and for DNA typing of individuals.

In addition, because of the large number of mitos in a cell, extracting mtDNA is easier from small amounts of tissue - and it can be readily separated form other DNA by centrifugation on CsCl gradients.

Human Evolution and mtDNAHuman Evolution and mtDNA

Extrapolating this in evolutionary terms, this means that all mitochondria came from a “single” ancestral female

- the so-called “Mitochondrial Eve”.References:

Proceedings of National Academy Sci (USA) 91:8739 (1994)Science 279: 28 (1998)

However, this is based on the assumption that mitochondrial inheritance is strictly clonal. Recent evidence shows that mitos from sperm do enter the egg and last for several hours. If recombination occurs between mitos, then the Eve hypothesis may be incorrect - or at least the timing would be incorrect.

Proc. R. Soc. Lond. B (1999) 266, 477-483

But we have to be careful: the rate of change in mtDNA may not be constant and heteroplasmy (due to recombination of mtDNA) may cause complications. Also, mtDNA represents a single lineage and other genetic changes need to be traced also.

Human Evolution and mtDNAHuman Evolution and mtDNA

However, when this was done with polymorphisms in the Y chromosome, ‘Adam’ was also traced back to Africa, at about the same period.

Assuming that the rate of change in the D-loop is constant and due only to mutation, the number of difference s between Africans can be use to calculate when their common ancestor lived. This works out to be about 200,000 years ago.

Human Evolution and mtDNAHuman Evolution and mtDNA

This suggests that modern Homo sapiens came out of Africa at about that time and migrated through Europe and Asia, replacing other early humans