Gene Activity: how genes work (part 1). Early 1900s Garrod suggests relationship between genes and metabolic disorders. “inborn error of metabolism” Garrod.

CHAPTER 14

Gene Activity: how genes work(part 1)

Early 1900s Garrod suggests relationship between genes and metabolic disorders. “inborn error of metabolism”

Garrod and others link genes to proteins.

1940 Beadle & Tatum: Neurospora crassa, bread mold experiment.

1949 Pauling & Itano’s Hemoglobin Experiment.

1950s Dr. Ingram shows structural differences in hemoglobin proteins.

Hemoglobin is a DIMER. Research showed only the beta chain is mutated. This lead to a revision of “one gene – one protein” to “one gene – one polypeptide”… (which is still not entirely true…)

BEADLE & TATUM

Step 1: grow spores in ‘enriched media’

had all necessary vitamins, amino acids, etc.

Step 2: blast ‘em with X rays

Step 3: grow spores on ‘minimal media’

had no vitamins, only sugars and salts

the mold would have to synthesize all of what it needs itself.

Wildtype mold can grow on this media, which means it’s fine making everything it needs.

Some of the mutant molds could NOT grow on this media, indicating they had a genetic defect in making a particular metabolite.

BEADLE & TATUM

Step 4:

BEADLE & TATUM

They did this for 2,000 strains.

Yeah.

BEADLE & TATUM

Of these, they found 3 strains.

The first couldn’t make vitamin B6

The second couldn’t make vitamin B1

The third couldn’t make para-aminobenoic acid.

BEADLE & TATUMIf the mutant that couldn’t make B6 was grown in minimal media

+ B6, it was able to grow JUST as well as the wildtype mold.

Additional work:

Simple genetic crosses showed a mendelian-like pattern of inheritance, fitting the theory that there was a single affected gene.

“Genes may control or regulate specific reactions in system, either acting directly as enzymes or determining the specificities of enzymes"

BEADLE & TATUM

Genetic analysis showed that each mutant differed from the original, normal type by only one gene. Biochemical studies showed that the mutants seemed to be blocked at certain steps in the normal metabolic pathways. Their cells contained large accumulations of the substance synthesized just prior to the blockage point - just as Garrod's patients had accumulated alkapton (link)

Their original paper

Another link that describes the experiment

BEATTLE & TATUM

(healthy would grow!)

PAULING & ITANO1. Purify Hemoglobin from

(a) healthy people (b) Homozygous recessive(c) Heterozygous

2. Run proteins on a gel3. Look at how they separate.

(more negatively charged)

(less negatively charged)

healthyheterozygous

Homozygous recessive

HbAHbA HbsHbs HbAHbs

(AND INGRAM)

Sickle Cell anemia was an autosomal recessive disorder that displayed incomplete dominance

GAG to GUG

This is normally a benign mutation, causing no apparent effects on the secondary, tertiary, or quaternary structure of hemoglobin. What it does allow for, under conditions of low oxygen concentration, is the polymerization of the HbS itself.

Because hemoglobin has 2 chains (alpha and beta), and only the beta chain is affected in sickle cell anemia, that means one gene controls for one polypeptide – not just an enzyme.

HOW GENES WORK: OVERVIEW

Genes code (somehow) for polypeptides.

Recall polypeptides are strings of amino acids (there are 20 amino acids)

DNA does NOT directly make polypeptides.

Instead, it has its information passed to a temporary message that is made of RNA.

The RNA leaves the nucleus for the cytoplasm.

In the cytoplasm, RNA attaches to ribosomes, which then read the message and produce a polypeptide.

RNA

• Differences between DNA & RNA• RNA uses ribose; DNA uses deoxyribose (draw

it out)• RNA uses the bases adenine, uracil, guanine,

and cytosine• Uracil replaces thymine.

• RNA does NOT form a double helix, but CAN produce single-stranded structures (example: “hairpin loop”)

MORE ON RNARNA has many other uses beyond acting as DNA’s messenger.

rRNARibosomes have rRNA built into them. This helps them read both

languages (nucleic acid + protein)

tRNAThere are specific tRNAs for each and every of the 20 amino acids.

Only when an amino acid is attached to a tRNA can it be used by a ribosome for protein synthesis.

Remember that RNA is made from a gene. This means that in addition to protein-encoding genes, there are also genes that code for rRNA and tRNA!

BREAKING THE CODE

There are 4 bases in DNA.

There are 20 amino acids in proteins.

How does the language of DNA translate into that for proteins?

In 1961, Nirenberg & Matthei broke the code.

They performed in vitro transcription experiments

THE LANGUAGE OF NUCLEIC ACIDS

Rules:

1. All words are 3-letters long and called “codons”.

2. A codon specifies either 1. a specific amino acid

2. “start translation”

3. “stop translation”

3. Some codons mean the same thing. (UUA and UUG both mean “leucine”)

THE LANGUAGE OF NUCLEIC ACIDS• Degenerate• Multiple words mean the same thing. There is more than one codon for most amino

acids.• ‘wobble effect’

• Unambiguous• Each codon has only a single meaning. (A word can’t mean more than one thing)

• Start & stop signals (1 start, 3 stops)• AUG = “start translation” (also, “methionine)• UAA, UGA, UAG = “stop translation”•

• Code is Universal, with some exceptions• Words mean the same thing for ALL organisms (this supports the laws of evolution

showing we all come from a common ancestor – our cells speak exactly the same complex language)

FYI

• The genetic code is, for the most part, universal, with few exceptions: mitochondrial genetics includes some of these. For most organisms the "stop codons" are “UAA”, “UAG”, and “UGA”. In vertebrate mitochondria “AGA” and “AGG” are also stop codons, but not “UGA”, which codes for tryptophan instead. “AUA” codes for isoleucine in most organisms but for methionine in vertebrate mitochondrial mRNA/tRNA.

THE WOBBLE EFFECT

The last letter of a codon is changed, but the amino acid it stands for does not.

UUA, UUG = both stand for leucine

GGU, GGC, GGA, GGG = glycine

WHY?

A protection against mutation. If the last base is mutated, the word’s meaning stays the same.

These kinds of mutations are called “silent”.

Protein

Nucleic Acids

Lipids

Carbohydrates

paradigm of molecular biology

polypeptide

ACDEFGHIKLMNPQRSTVWY

nucleic acids

A

T

C

G

T

A

G

C

RNA

A

U

C

G

U

A

G

C

transcription

translation

Transcribing fixed, permanent information onto a portable, temporary templateTranslating language of DNA into language of protein

folding

STRUCTURE&

FUNCTION

DNA

mature protein

TRANSCRIPTION

DNA serves as a template to make mRNA.

All 3 kinds of RNA are formed this way.

We will only focus on the one that makes a protein.

TRANSCRIPTION• tRNA, rRNA, and mRNA are all created through transcription. Since

mRNA encodes for protein information, we will deal with this exclusively.

• Promoter• Determine whether a gene is “on” or “off”.• Dictates where RNA polymerase binds, and which strand to

read.

• DNA: Coding strand = sense strand; noncoding strand = antisense strand.

• DNA:DNA is much stronger than DNA:RNA binding• 5’ to 3’.

• Step 1: Initiation, elongation, termination (stop codon is ‘read’)

Step 2: mRNA is processed (cap, tail, splicing)– Introns vs. exons

Step 3: the ‘mature’ mRNA exits the nucleus.

Next up: Translation

TRANSLATION

• After transcription, we have:

AAAAAAAAAAAAAAAA

5’ Cap 3’ poly-A tailSpliced transcript (exons only!)

TRANSLATION

• The mRNA goes into the cytoplasm.• It attaches to the small subunit of the

ribosome, and the ‘initiator’ tRNA (which is attached to a methionine)

• Movie 1• Movie 2• Movie 3 (advanced)

TRANSLATION• TYPO: page 245. Transcription should be TRANSLATION! Ugh.• Step 1: Initiation

– Initiation factors bind to the cap of the mRNA. – The “start” codon is recognized. The small ribosomal subunit attaches.– Initiator tRNA (tRNA-methionine) binds to its codon.– The large ribosomal subunit attaches.

• Step 2: Elongation– New tRNA-amino acid molecules (“aminoacyl tRNAs”) floating around bind to their

codons.– The Ribosome machine adds the new amino acid to the growing chain (in the “P” site for

peptide site).– The tRNA that was in the “P” site moves to the “E” site (exit), while the tRNA that was in

the A site (for “amino acid”) moves over to the “P” site.• In e.coli this is about 15 codons read per second.

– The growing chain is folding into a 3D structure as it is build. Sometimes, “chaperone” proteins help the folding process.

• Step 3: Termination– When a termination codon is read by the ribosome, a “release” factor binds to the A

site. This causes the whole machine to fall apart.

GENE: THE DEFINITION• Definition of a mutation:

– Used to be something that causes a change in phenotype.– Now we know it’s a change in the DNA sequence of a gene.

• What’s a gene?– Think of it as simply a sequence of DNA that gives rise to a piece of

RNA.• Genes that encode for mRNA (which code for proteins) are

called “protein coding genes”• Genes that encode for tRNA and rRNA are called “noncoding”

genes.• There are also genes that encode pieces of RNA that bind to

mRNA… inhibiting them from being translated. This is called RNA inhibition, or simply RNAi. This is a new world of research, and one of the largest areas of new work today.