biol 1030 introduction to biology: organismal biology...

BIOL 1030 Introduction to

Biology: Organismal Biology. Spring 2011

Section ASteve Thompson: stthompson@valdosta.edu

http://www.bioinfo4u.net1

Saturday, February 19, 2011

DNA transcription and gene regulation

We’ve seen how the principles of classical genetics affects the heredity of phenotypic

traits, and we’ve seen how DNA is duplicated and segregated in mitosis and meiosis, but where do

all those proteins that make up a phenotype come from, and how do they end up in the right

place at the right time?!Nice series at Wikipeia: http://en.wikipedia.org/

wiki/Gene_expression.2

Remember DNA’s structure!It’s a double helix, where the . . .“Rungs” are base pairs joined by hydrogen bonds. And the bases . . .Adenine (A) pairs with thymine (T), and . . .Cytosine (C) pairs with guanine (G).The strands are complementary, and . . .They are oriented in opposite directions,5’ to 3’, or 3’ to 5’.

3Saturday, February 19, 2011

This should be very familiar to you by now!

Biology’s “Central Dogma”DNA➔RNA➔proteinTranscription – cell copies DNA to RNA.Translation – RNA information is used to manufacture proteins.

In Eukaryotes transcription happens inside the nucleus, whereas translation happens outside it.

Here’s an overview.

http://www.valdosta.edu/~stthompson/animations/Chapter12/gene_expression.swf6

Remember the two types of nucleic acid: DNA and RNA.

Not structural and catalytic RNA

And the way the base pairing works.

We’ll cover three types of RNA today:

1. Messenger RNA (mRNA) — carries information specific to a protein; three RNA (or DNA) bases form a “codon,” specifying a particular amino acid.

2. Ribosomal RNAs (rRNA) — combine with proteins to form a ribosome.

3. Transfer RNA (tRNA) — carries a specific amino acid to the ribosome.

One more time . . .

Transcription (DNA to RNA) has three major steps:

1. Initiation —a) Enzymes unwind DNA exposing template strand;b) RNA polymerase binds to the promoter.

2. Elongation —a) RNA polymerase moves 3’ to 5’ along strand.

3. Termination —a) RNA polymerase reaches the terminator

sequence at the end of the gene;b) RNA separates – may be mRNA, tRNA, or rRNA;c) DNA reforms its helix.

Here are the steps of

transcription illustrated,

broken down into the three major events.

And here it is in animation.

http://www.valdosta.edu/~stthompson/animations/Chapter12/stages_transcription.swf

mRNA modification . . .In Bacteria and Archaea, ribosomes begin translating mRNA as soon as transcription is complete. This all happens in the cytosol of the cell (cytoplasm).In eukaryotes mRNA is usually modified after transcription.A 5’ cap and a poly-A tail is added to enhance translation by helping the ribosome to attach. Why and how? It all seems to relate to export from the nucleus and mRNA stability. See http://en.wikipedia.org/wiki/5'_cap and http://en.wikipedia.org/wiki/Poly_a_tail for simple expanations.And the biggy — introns (intervening sequences) are removed, leaving exons, which are spliced together by spliceosomes (complex factories of structural and catalytic RNAs and associated protein that do just this splicing job; see http://en.wikipedia.org/wiki/Spliceosomes).

mRNA modification occurs in the nucleus in Eukaryotes.

Contrast this with Bacteria and Archaea.

http://www.valdosta.edu/~stthompson/animations/Chapter12/process_gene_information.swf

On to translation – mRNA to protein.

The ‘universal’ genetic code: An mRNA codon with three bases specifies an amino acid, and

signals start and stop. But it’s redundant!

Translation requires all three major types of RNA.

1. mRNA – contains the genetic information specifying an exact amino acid order in the codons encoded in an organism’s DNA.

2. tRNA – specific tRNAs bring specific amino acids to the ribosome by pairing their anticodon to each mRNA codon.

3. Ribosome – a combination of rRNAs and their associated proteins that make little protein manufacturing ‘nano-factories.’

The tRNA has a clover-leaf shape with the amino acid site on the ‘stem.’

The anticodon is on

the tRNA molecule!

The ribosome has two major components.

Here’s an example from a Eukaryote.

It contains 82 proteins and four rRNAs (http://

www.facstaff.bucknell.edu/kfield/

organelles/ribosome.html).

Translation also has three steps:1. Initiation:

a) The mRNA start codon binds to the small ribosomal subunit.b) The First tRNA (methionine) binds to the mRNA codon.

2. Elongation:a) The large ribosomal subunit attaches to the complex.b) The tRNA corresponding to the second codon attaches.c) A covalent bond (the peptide bond) is formed between

adjacent amino acids.d) The ribosome releases the empty first tRNA.e) The ribosome shifts down one codon, allowing the third

tRNA to bind.f) The polypeptide grows one amino acid at a time.

3. Termination:a) The stop codon (UGA, UAG, or UAA) is reached, and the . . .b) New polypeptide is released.

Here’s an overview, starting with initiation.

This all happens both ‘free’ in the cytosol and bound on the outside of

the ER membranes in

Eukaryotes (and in mitochondria

and chloroplasts).

And then the large subunit

binds and elongation

starts.Saturday, February 19, 2011

And finally a stop codon is reached

and termination

occurs.Saturday, February 19, 2011

Here’s a nice overview.

http://www.valdosta.edu/~stthompson/animations/Chapter12/translation.swf

The chain grows and

multiple proteins are built at the same time.

Sometimes chaperones help.

And one more time, the whole process.

http://www.valdosta.edu/~stthompson/animations/Chapter12/protein_synthesis.swf

Protein folding . . .Proteins must achieve their final functional shape before they can work – some regions attract or repel, enzymes catalyze bonding, “chaperone” proteins may stabilize the process.Errors in folding can lead to illness, e.g. prions.Some proteins must be altered. For example . . .Insulin has a bunch of amino acids removed.Many proteins don’t work alone (quaternary structure). For example . . .Hemoglobin has four separate polypeptide subunits (two alpha and two beta in adult).

Regulation – control of gene expression:Protein synthesis is fast and efficient, but it has a . . .Tremendous ATP requirement. For example E. coli spends 90% of it ATP on protein synthesis. Therefore, . . .Cells save energy by not producing unneeded proteins (plus differentiation).Only those proteins that are needed by that particular cell at that particular time are produced.

Operons in bacteria . . .Operons are a group of genes plus their promoter and operator, which control the transcription of the group as a whole.Promoter – where RNA polymerase attaches.Operator – DNA sequence where a repressor can bind to inhibit transcription.For example, the lac operon – three lactose degrading enzymes plus their promoter and operator.Without lactose a repressor turns genes off.With lactose, a modified form of the sugar binds to the repressor causing it to fall off the promoter. Transcription can proceed.

Here’s how the components are organized on the

bacterial DNA.

And here’s what happens without lactose around.

But with lactose in the environment . . .

Eukaryotes are way more complex!Different cell types must express different subsets of genes at different times (this is known as cell differentiation).Transcription factors (>2600 known in humans) are required for RNA polymerase to bind to a promotor, See e.g. the EPD database, http://www.epd.isb-sib.ch.Signal transduction is one way of activating and/or deactivating various transcription factors.And there’s a slew of additional regulatory mechanisms, e.g. . . .

Methylation, siRNAs, miRNAs and . . .Alternative splicing. Plus . . .

Many, many others!35

Here’s a way oversimplified view of how a transcription factor works.Many databases of these exist, e.g. http://dbd.mrc-lmb.cam.ac.uk/DBD/index.cgi?

Home and http://jaspar.cgb.ki.se/

And here’s an example of alternative splicing.

This is partly how the 21,000 some human genes can encode a million or so different proteins!

And now for something completely different: Mutations . . .

Are any change in a cell’s DNA sequence from its original sequence.It happens during DNA replication, and can be good, bad, or silent (neutral).There are many types of mutation: One is a “point” mutation. This substitutes one DNA nitrogenous base for another (this is known as a single nucleotide polymorphism, SNP).If it occurs in an exon, it can be “silent” (synonymous), if the same amino acid is specified. Or it . . .Can be “nonsense” if it changes a codon into a “stop.” Or it . . .Can be “missense” (nonsynonymous) if it changes the amino acid, which, again, can be good, bad, or neutral.However, it may cause disease, e.g. sickle cell anemia (but this is a mutation that is inherited, i.e. it is in every cell of your body).

Most forms of sickle cell

are a one amino acid

change caused by a single base

substitution. But you

inherit this mutation, it doesn’t occur in your blood!

Here’s an example of a single point substitution (SNP)

http://www.valdosta.edu/~stthompson/animations/Chapter12/Mutation_by_base_substitution.swf

Another class of mutations are base insertions and deletions.If these occur in an exon, and are not in multiples of three, then they will cause a . . .Frameshift mutation, which will almost always destroy the encoded protein!An expanding repeat is another type — the number of copies of a particular nucleotide sequence increases over several generations. If this occurs in an exon, and is in multiples of three, then it will cause an amino acid repeat sequence to expand, e.g. some types of Huntington’s chorea.Insertions and deletions can occur in crossing over during meiosis. Therefore, the progeny will have ‘em.

Here’s an example of what happens

when you insert extra

bases into an exon (reading frame).

An animation illustrates a simple insertion or deletion mutation that can occur during DNA replication.

http://www.valdosta.edu/~stthompson/animations/Chapter12/Addition_and_deletion_mutations.swf

And here’s an example of an

entire gene getting

duplicated in one chromatid and deleted in

the other during

crossing over in meiosis. 45

Know the vocabulary!

However, remember “wild type” is a bit of a misnomer, and

I think nonsynonymous is way easier to remember than

“missense.” Plus,

synonymous, i.e. “silent” isn’t shown here.

A “Stop” was created.

Causes of mutation:Spontaneous – DNA replication errors can cause substitution (point), insertion, or deletion mutations.Meiotic error – crossing over mistakes can cause duplication or deletion mutations, and . . .Chromosome inversions or translocations.Transposons – are moveable, “jumping gene” DNA sequences that can insert themselves into a reading frame, interrupting its function.Mutation can be accelerated by mutagens – external agents, e.g. radiation, and many different chemicals, e.g. ‘tar’ in cigarettes!

Heritable mutations versus . . .Somatic mutations, which occur in nonsex cells, i.e. all the cells of your body except those that create sperm or eggs through meiosis. Those are called “germ” cells.All cells derived (through mitosis) from a mutated somatic cell carry that mutation (e.g. cancer).The mutation is not passed to any offspring!Contrast this with a germline mutation. It is . . .Heritable, and will passed on in every gamete produced by that individual. Plus it will be passed on for every generation from that point on (according to the odds of classical genetics as illustrated with Punnett squares).

Mutations are incredibly important! As they . . .Are the source of new gene variants (alleles) that fuel evolution; e.g. . . .Random mutations can result in antibiotic resistant bacteria, which ‘sweep’ a population, which this has become a huge problem in health care.And they also provide the initial variation that sex can amplify.

OK — don’t space it out . . .

We have our second exam next class meeting! It will cover

everything since Exam I, in other words, all of classical and

molecular genetics.50

biol 1030 introduction to biology: organismal biology...

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