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The Molecular Basis of Inheritance
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By the 1940’s, scientists knew that chromosomes carried hereditary material and
consisted of DNA & protein.
Two experiments proved that it was DNA only.
Frederick Griffith (1928)
- He was trying to find a vaccine against Streptococcus pneumonidae (bacteria that
causes pneumonia in mammals).
- Used two strains of bacteria ‘S’ and ‘R’.
- Showed that bacteria could undergo transformation: bacteria taking up naked
DNA for the surrounding environment.
Avery (1944)
- Performed Griffith’s experiments and took it even farther.
- Concluded DNA was the hereditary material, but was still met with skepticism.
Hershey & Chase (1952)
- Discovered that DNA was the genetic material of a phage (virus) known as T2.
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Watson & Crick (1953)
- Discovered that DNA was a double helix.
- The helix was ladder-like with a sugar-phosphate backbone.
- The 2 backbones were antiparallel; they ran in opposite directions.
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There are 4 nitrogenous bases:
- Adenine (A) purine
- Guanine (G) purine
- Thymine (T) pyrimidine
- Cytosine (C) pyrimidine
Purines bond with pyrimidines.
- A = T (2 bonds) and C = G (3 bonds)
Since A always bond with T, their amounts in a strand of DNA are equal (same for GC).
Weak hydrogen bonds hold the two strands of DNA together.
DNA replication
- There are two strands that both need to be replicated.
- Before replication, these strands must be separated.
- Once separated, these strands act as the template for assembling a complementary
strand.
- 3 Hypotheses for Replication:
- 1. Conservative
- The parental double helix should remain intact and the 2nd (new ) double
helix is made from entirely new material.
- 2. Semiconservative
- Each of the 2 resulting DNA molecules are composed of one original template
and one newly created strand.
- 3. Dispersive
- Both strands of each new helix contain both a mixture of old and new DNA.
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DNA replication is done in the semiconservative fashion; the other 2 hypotheses are
not correct.
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The process of DNA replication is:
- Complex: The helix untwists as it copies its two antiparallel strands
simultaneously. This requires the cooperation of over a dozen enzymes &
proteins.
- Extremely Rapid: In prokaryotes, up to 500 nucleotides are added per second. It
takes only a few hours to copy the 6 billion bases of a single human cell.
- Accurate: Only about one in a billion nucleotides are incorrectly paired.
Replication must start at specific sites. They are called the origins of replication.
These origins have a specific nucleotide sequence.
Specific proteins must bind to the origins to initiate replication.
In addition to proteins at the origin, a primer is needed to “prime” the reaction.
Primer: short RNA segment that is complementary to a DNA segment.
Primers are short segments of RNA polymerized by an enzyme called primase.
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The DNA helix opens up at an origin and a replication fork is created.
DNA helicase is responsible for opening up the fork.
The forks spread in both directions away from the central initiation point creating a
replication bubble.
Prokaryotic cells (and viral DNA) only have one origin.
Eukaryotic DNA has many origins creating many forks = many bubbles.
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Enzymes called DNA polymerases (DNA pol) catalyze the synthesis of a new strand.
DNA pol links the nucleotides to the growing strand.
ALL DNA must be replicated in the 5’ to 3’ direction. (5’ → 3’)
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Continuous synthesis of both DNA strands at a replication fork in the same direction
is not possible because DNA pol replicates 5’ → 3’
The problem is solved by the continuous synthesis of one strand, the leading strand,
and discontinuous synthesis of the lagging strand.
The lagging strand
- It is produced as a short series of segments called Okazaki fragments which are
individually made in the 5’ → 3’ direction.
- Okazaki fragments are 1,000 to 2,000 nucleotides long in prokaryotes and 100 to
200 long in eukaryotes.
- The lagging strand has many RNA primers.
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When the lagging strand is complete, RNA primers are removed by DNA pol and
replaced with DNA.
Then they are linked together by an enzyme called DNA ligase.
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Enzymes proofread DNA during its replication and repair damage in existing DNA.
Mismatch Repair
- Corrects mistakes when DNA is synthesized.
- DNA pol and other proteins assist in this process.
- A heredity defect in one of these proteins has been found with one form of colon
cancer.
- In the absence of proofreading, errors accumulate.
Excision Repair
- Corrects accidental changes that occur in existing DNA.
- Changes can result from UV light, cigarette smoke, etc.
- There are more than 50 types of DNA enzymes that repair damage.
Transcription & Translation
- A gene is a segment of DNA that codes for a protein product.
- RNA links DNA’s genetic instructions for making proteins.
- They are the two main steps that form a gene to a protein.
- Transcription: Making RNA from DNA
- Translation: ‘translating’ the RNA into a protein
- Remember that RNA is different from DNA in that:
- It is single stranded.
- Has ribose instead of deoxyribose
- Has Uracil instead of thymine
- The linear sequence of nucleotides (A, C, G, T) in DNA ultimately determines the
linear sequence of amino acids in a protein (the primary structure).
- Genes can be hundreds or thousands of nucleotides long.
Transcription
- Genes are transcribed from DNA into mRNA.
- DNA is read as codons.
- Codon: is 3 nucleotides long and codes for a specific amino acid.
- Codons are the “words”; when the amino acids they make are linked together, they
make the sentence (protein).
- Genes are not directly translated into amino acids, but first are transcribed as codons
in mRNA.
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DNA is a double helix, however, only 1 of these strands is transcribed.
The strand that is transcribed is called the template strand.
The other strand is NOT transcribed and is called the non-template strand; it serves as
a template for making a new strand during replication.
An mRNA will be complementary to the DNA template from which it is transcribed.
- Ex: if DNA reads “CCG” then the RNA that is made will read “GGC”
- Try: DNA reads “CAT”
- Then: RNA reads “GUA”
Similar to DNA replication (RNA primer), there is a promoter region that is required
to start the production of mRNA.
RNA pol will bind to this promoter region (~ 100 nucleotides long).
RNA pol cannot recognize the region without the help of transcription factors.
TATA box: A short nucleotide sequence at the promoter region that is T and A rich.
- The box is ~ 25 nucleotides ‘upstream’ from the initiation site.
Once transcription begins, RNA pol II moves along DNA and performs 2 functions:
- 1. It untwists & opens a short segment of DNA exposing about nucleotides.
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2. It links incoming RNA nucleotides to the 3’ end of the elongating
strand. Therefore, RNA grows one nucleotide at a time in the 5’ → 3’ direction.
As the mRNA strand elongates, it peels away from the DNA template.
- It can grow ~ 30-60 nucleotides per second.
Transcription continues until it reaches a termination sequence.
- In eukaryotes, the most common sequence is ATAAA.
Translation
- The synthesis of a polypeptide, which occurs under the direction of mRNA.
- The mRNA made in transcription leaves the nucleus and then travels into the
cytoplasm to be translated.
- Translation occurs on the ribosomes.
- Ribosomes are made of rRNA
- Ribosomes facilitate the orderly linking of amino acids into polypeptide chains.
- During translation, the linear sequence of codons along mRNA is translated into the
linear sequence of amino acids in a polypeptide.
- Because codons are triplets, the # of nucleotides making up a polypeptide is 3 times
the # of amino acids.
- By the 1960’s, all 64 codons were decoded.
- 61 out of 64 code for amino acids.
- The triplet AUG has two functions: start and Methionine.
- Three codons code for signal termination (stop): UAA, UAG, & UGA
- There is redundancy in the genetic code, but no ambiguity.
- Redundancy exists because 2 or more codons can code for the same AA.
- Ex: UUU & UUC both code for the amino acid Phenylalanine (Phe)
- There is no ambiguity: each codon can only code for one type of amino acid or each
codon can only do one specific thing
- Reading Frame:
- The correct grouping of nucleotides is important in the molecular language.
- Sequences of amino acids are only produced if the correct codons are grouped.
- Changes in the reading frame could have no effect or drastic effects.
- THE CAT ATE THE RAT.
- Adding one letter could change the meaning.
- THE CAT SAT ETH ERA T.
- The genetic code is universal:
- Among every living organism, each codon means the same thing.
- Ex: UUG codes for Trp in humans, slugs, flies, fish etc.
- There are some minor exception in ciliates (group of Protists)
- For translation to occur, there must be 3 things present at the same location:
- 1. mRNA (has the DNA code)
- 2. rRNA (the ribosome)
- 3. tRNA (transfers each amino acid to a growing change)
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tRNA
- It aligns the appropriate AA’s to form a new polypeptide.
- It contains anticodons which have the opposite sequence of the mRNA codon;
therefore, they bind.
- Each tRNA has the correct AA for each anticodon.
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Ex of anticodons & tRNA
- The mRNA codon UUU is translated as the AA phenylalanine (Phe).
- The tRNA that transfers Phe to the growing chain has an anticodon of AAA.
The ability of tRNA to carry the specific AA for its anticodon depends on its
structure.
Is only about 80 nucleotides long.
A loop protrudes at one end where the anticodon is located.
At the other end (3’ end) is the attachment site for the correct AA.
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Exon: codes for proteins
Introns: no protein product; “junk” DNA
Eukaryotes have more introns than exons.
Introns:
- May control gene activity.
- Play a role in the evolution of protein diversity; they increase the probability that
recombination (crossing over) will occur between alleles.
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Mutations
- Any permanent change in DNA that can involve large chromosomal regions, or a
single nucleotide pair.
- Point Mutation: limited to one nucleotide in a single gene pair.
- 1. Base Pair Substitution
- The replacement of one base pair with another.
- Occurs when a nucleotide and its partner from the complementary DNA
strand are replaced with another pair of nucleotides.
- Depending on how base-pair substitutions are translated, they can result in
little or no change in the protein encoded by the mutated gene.
- Redundancy in the genetic code is why some substitution mutations have
no net effect.
- A base pair change may simply transform one codon into another that
codes for the same AA.
- The substitution may occur in an intron.
- Some mutations can have drastic effects.
- Sickle cell anemia
- One AA is changed due to one nucleotide being changed.
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Mis-sense mutation: substitution that alters an AA codon
Non-sense mutation: a substitution that goes from coding an AA into a
stop codon.
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Insertions or Deletions
Usually have a greater negative effect on proteins than substitutions.
Inserting or deleting one single nucleotide will alter the reading frame.
This is called a frameshift mutation.
This most likely will produce a non-f(x)’al protein.