DNA

Chapter 16

•Cell and organism characteristics are expressed through protein.

•Cell structures are composed largely of protein

•Membrane transport is governed by protein. •Pigments are protein. •Antibiotics are protein. •Hormones are principally protein. •Most importantly, enzymes are protein.

How do we know DNA controls genes?? Early 20th century experiments with bacteria

showed that a bacterial strain could be "altered" by injecting it with foreign DNA. The biology giant who pioneered this work in 1928 was Frederick Griffith, an English Physician.

Griffith was studying the bacterium Streptococcus pneumoniae

He was trying to develop a vaccine against the bacterium, because antibiotics were unknown.

Griffith worked with two strains of the bacteria, S and R.

S = shiny, smooth colonies - (the virulence of this colony is caused by a polysaccharide capsule that protects the bacterium from the defense mechanisms of the host.)

R = rough, unattractive colonies. - (no protection from host's antibodies because they lack a "slime coat")

When Griffith injected S strain into the mice, they died within 1 day (their little rodent hearts were found teeming with bacteria.)

When Griffith injected S strain that had been heat-treated (remember, he was trying to develop a vaccine) into the mice, they did NOT become diseased. (The heat influenced some "factor")

When Griffith injected R strain AND heat-treated S strain bacteria into the mice, to his astonishment, they died!!!!!!!

Griffith concluded that, in the presence of the "dead" S pneumococci, some of the inoculated R pneumococci had been transformed into virulent organisms. He called the phenomenon transformation.

•From Griffith's experiments, scientists of the day concluded that the living cells were "converted" by what they called "transforming factor" {Remember, they had no knowledge of DNA}

•Scientists also observed that the "transformed" colonies produced pathogenic descendents. Clearly, some inheritable factor was involved.

•We now credit Frederick Griffith with discovering transformation in bacteria and establishing the foundation of molecular genetics.

Griffith’s work led to a 14-year search for the “transforming factor”. The identification of the "transforming factor" was accomplished by Oswald T. Avery at Rockefeller University. He and his colleagues treated samples of the pathogenic heat-killed bacteria in a variety of ways to destroy different types of substances - proteins, nucleic acids, carbohydrates, and lipids. They then tested what was left to see if it had retained transforming activity. They found that if DNA was destroyed, transforming activity was lost. Everything else was dispensable. Avery and his colleagues Maclyn Macleod and Colin McCarty isolated DNA and showed that it caused the bacterial transformation.

•Avery (left) , MacLeod (middle) , and McCarty (right) published in 1944, but they were scorned by their colleagues. DNA was thought not to be "complex“ enough, and it was not even known that bacteria had genes.

Alfred D. Hershey (right) and Martha Chase (left) published a report in 1952 that finally established that DNA was the hereditary material. They injected bacterial cells with both proteins and DNA from phage viruses, and found that of the two, DNA (and not protein) "infected" the bacteria. By this time, genes were also known to exist in bacteria. The Hershey-Chase experiment established that DNA was the hereditary material in viruses.

Here is the evidence that all of these scientists determining DNA structure had to work with: X-ray studies of DNA provided clues about the

dimensions of DNA and hinted that its shape was helical. The most important of these x-ray analyses came from the English chemist Rosalind Franklin (left). Her work would have been impossible except for the success of English biophysicist Maurice Wilkins (right) in preparing uniformly oriented DNA fibers

This image hinted that the shape of DNA was a double helix (like 2 circular staircases), and part of the core of the helix was hollow. It also showed a concentration of matter on the outside. Additionally, the nitrogenous bases on the inside maintained a uniform width. Your view of this molecule is top down through the middle.

Much was also known about the chemical composition of DNA. It was known that: DNA is a polymer of nucleotides. Each nucleotide consists of the sugar deoxyribose,

a phosphate group, and a nitrogen-containing base. There exist four bases in DNA; adenine, thymine,

guanine, cytosine. Erwin Chargaff (Columbia University) determined

that in all living species, A = T, and C = G Chargaff's Rule) (1950).

Thus, the solution of the problem of “What is the shape of DNA?” was tied to three issues: What is the physical, 3-D form of DNA? How does DNA make copies of itself? How does DNA regulate protein synthesis?

Answers to the first two questions were provided by the famous pair Francis Crick (English physicist) and James Watson (American geneticist) who worked together at Cambridge University in England.

1953 2003

Watson and Crick combined all that had been learned so far about DNA structure into a coherent model (1953) and suggested a mechanism for replication. They hurriedly published their findings in a 2-page paper and were ultimately awarded Nobel Prizes (1956) for their huge contribution to our understanding of molecular genetics. James Watson currently heads the Human Genome Project, and Francis Crick passed away on July

30, 2004, the victim of cancer.

In the early 1950s, the race to discover DNA was on. At Cambridge University, graduate student Francis Crick and research fellow James Watson (b. 1928) had become interested, impressed especially by Pauling's work. Meanwhile at King's College in London, Maurice Wilkins (b. 1916) and Rosalind Franklin were also studying DNA. The Cambridge team's approach was to make physical models to narrow down the possibilities and eventually create an accurate picture of the molecule. The King's team took an experimental approach, looking particularly at x-ray diffraction images of DNA.

In 1951, Watson attended a lecture by Franklin on her work to date. She had found that DNA can exist in two forms, depending on the relative humidity in the surrounding air. This had helped her deduce that the phosphate part of the molecule was on the outside. Watson returned to Cambridge with a rather muddy recollection of the facts Franklin had presented, though clearly critical of her lecture style and personal appearance. Based on this information, Watson and Crick made a failed model. It caused the head of their unit to tell them to stop DNA research. But the subject just kept coming up.

Franklin, working mostly alone, found that her x-ray diffractions showed that the "wet" form of DNA (in the higher humidity) had all the characteristics of a helix. She suspected that all DNA was helical but did not want to announce this finding until she had sufficient evidence on the other form as well. Wilkins was frustrated. In January, 1953, he showed Franklin's results to Watson, apparently without her knowledge or consent. Crick later admitted, "I'm afraid we always used to adopt -- let's say, a patronizing attitude towards her."

Watson and Crick took a crucial conceptual step, suggesting the molecule was made of two chains of nucleotides, each in a helix as Franklin had found, but one going up and the other going down. Crick had just learned of Chargaff's findings about base pairs in the summer of 1952. He added that to the model, so that matching base pairs interlocked in the middle of the double helix to keep the distance between the chains constant.

Watson and Crick showed that each strand of the DNA molecule was a template for the other. During cell division the two strands separate and on each strand a new "other half" is built, just like the one before. This way DNA can reproduce itself without changing its structure -- except for occasional errors, or mutations.

The structure so perfectly fit the experimental data that it was almost immediately accepted. DNA's discovery has been called the most important biological work of the last 100 years, and the field it opened may be the scientific frontier for the next 100. By 1962, when Watson, Crick, and Wilkins won the Nobel Prize for physiology/medicine, Franklin had died. The Nobel Prize only goes to living recipients, and can only be shared among three winners. Were she alive, she would have been included in the prize.

The DNA molecule is: (1) a double-stranded helix (2) of uniform diameter (3) that twists to the right (like a screw) (4) with two strands running in opposite directions (i.e. it is “anti-parallel”)

The sugar-phosphate "backbone" coils around the outside of the molecule, and the nitrogenous bases are found on the "inside" of the double-helix, like steps on a ladder.

The two chains are held together by hydrogen bonding between specifically paired bases: A with T, and C with G.

Adenine pairs with Thymine by forming two hydrogen bonds.

Guanine pairs with cytosine by forming three hydrogen bonds.

Every base pair consists of one purine (A or G) and one pyrimidine (T or C)

This purine-pyrimidine pairing insures a constant internal diameter.

What do we mean when we say that the two DNA strands run in "opposite directions"?

The "direction" of a nucleotide is defined by looking at the phosphate-sugar linkages on the outside of the molecule.The “last” Carbon is the 3’ Carbon

The phosphate groups connect to the 3' carbon of one deoxyribose molecule and the 5‘ carbon of the next, linking sugars together.

DNA has a free 5' phosphate at one end - the 5' end - and a free 3' hydroxyl (-OH) group at the other - the 3' end. Thus, we say that this "side" runs in the 5' ->3' direction. The 5' end of one strand in a DNA double helix is paired with the 3‘ end of the other strand, and vice versa; that is, the strands run in opposite directions. The molecule is “anti-parallel”.

There were three models for DNA Replication. All 3 obeyed base pairing rules, but only 1 model proved to be correct: The Semiconservative Model

How does the enzymatic machinery of the nucleus replicate DNA semiconservatively? Each DNA strand must function as a "template" to build a

new, complimentary strand. The A-T and C-G base pairing rules must be followed. Free A, T, C, and G nucleotides must be available. There must be a DNA polymerase to assemble the DNA

molecule. There must be a source of energy to drive this endergonic

reaction

A key note: DNA nucleotides are always added to the newly assembled strand at the 3' end. (ie. Assembly occurs in the 5' to 3' direction)

The enzyme that attaches one DNA nucleotide to the next in the 5’ to 3’ direction is named DNA polymerase.

Before replication can begin, the two strands must be "unwound" and separated. Enzymes called helicases unwind (“unzip”) the double helix.

Topoisomerase is anenzyme that cuts and rejoins nucleotide linkages at the site of the phosphate so that the double helix may be Topoisomerase is an temporarily “unwound” during replication

Prokaryotic replication begins at one site on the circular strand of DNA and proceeds at both ends until it meets on the "other side".

Eukaryotic replication occurs at many sites along the length of the molecule - otherwise it would "take too long" (Eukaryotic DNA is extremely long)

The site where DNA is separating into two strands is a "replication fork"

The replication forks are moving, Y-shaped structures. It is here that new DNA strands are synthesized by adding DNA nucleotides.

Note: DNA polymerase CAN add DNA nucleotides to a polynucleotide strand, but it cannot start a strand from scratch.

At every new replication fork in eukaryotic cells (and at the beginning of every Okazaki fragment {more later}), the enzyme primase is used to bind to DNA and synthesize an RNA primer. Then, DNA polymerase can take over and complete the synthesis of the complimentary strand. In DNA replication, the primer is a short single strand of RNA. This RNA is complimentary to the DNA template strand.

Once the primers have been formed, DNA replication continues. There are two strands in the replication fork, a "leading strand" and a "lagging strand". The leading strand grows when DNA polymerase adds nucleotides, one at a time, in the 5' to 3' direction.

The other strand, the "lagging strand" presents a problem because the rule states that "DNA nucleotides can only be added in the 5' to 3' direction". But, as the diagram to the right shows, the end of the "lagging strand" closest to the replication fork is the 5' end, and

new nucleotides cannot be added here.

Nature’s solution to this problem is to assemble small, complimentary sections of DNA in the 5’ to 3’ direction, and then fuse them. These “small, complimentary sections” are called Okazaki Fragments.

Okazaki fragments are synthesized along the DNA template, in the 5' to 3' direction, one nucleotide at a time.

Okazaki fragments are from 100-200 nucleotides long in eukaryotes.

Once the Okazaki fragments are synthesized (in the 5' to 3‘ direction), DNA ligase joins adjacent fragments to elongate the replicant copy of DNA on the lagging strand.

Here's how the complimentary lagging end is formed: RNA primase forms an RNA primer - DNA strands will form

only from the 3' end of the primer. DNA nucleotides are added to the 3' end of the primer, until

they meet the Okazaki fragment that was synthesized immediately before.

The primer of the older Okazaki fragment is hydrolyzed, and the new Okazaki fragment is extended to join the old one. The two are joined by the enzyme DNA ligase.

Eventually, the entire strand of DNA separates as replication bubbles join with one another. The very last part of the DNA molecule to separate is the centromere, which holds the two complimentary sister strands together until Anaphase.

http://www.youtube.com/watch?v=yqESR7E4b_8http://www.youtube.com/watch?v=AJNoTmWsE0s

This is the unit that deals with what DNA "does", not "what it is".

•What DNA "does" is oversee (direct) Protein Synthesis. In simpler words, DNA tells the cell what types of protein to make.

•"One gene, one protein" (Better stated: "One gene, one polypeptide", but it doesn't rhyme.)

The information encoded in DNA is first transcribed to RNA.

RNA and a team of proteins then translate this encoded message into a new protein molecule.

The protein is then altered, and comes to perform its final function (note: most likely as an enzyme - the greatest # of genes encode for enzymes.)

Errors can occur during transcription or translation. These errors manifest themselves as mutations.

Genotype - a description of the genes possessed by an organism.

Phenotype - a description of an organism's appearance. What we are really describing, then, is a series of

chemical reactions that begin with a nucleotide sequence in DNA and end with the synthesis of a particular polypeptide.

There is, then, a relationship between the nucleotide sequence in DNA and the amino acid sequence in a polypeptide. (3:1 Remember For Later 3:1)

RNA is a polynucleotide similar to DNA but different in at least 3 ways:

RNA consists as only one polynucleotide strand (ie. It is single-stranded) {Chargaff's Rule is true only for DNA}

How RNA Differs From DNA

The sugar molecule found in ribonucleotides is ribose rather than deoxyribose

Three of the nitrogenous bases (A,C, and G) in RNA are the same as DNA, but the fourth is different. Uracil is substituted for Thymine. The structural difference between the two is minute: uracil lacks a methyl (-CH3) group

In most organisms, DNA stores all the cell's genetic information, while RNA is a temporary messenger molecule. (Exception: retroviruses)

RNA can base-pair with single-stranded DNA, but not with closed, double-stranded DNA. This pairing obeys the A-T, U-A, and G-C hydrogen-bonding rules.

RNA can also fold over and base-pair with itself!

Francis Crick proposed the Central Dogma of molecular biology: DNA codes for the production of RNA (during transcription), and RNA codes for the production of protein (during translation).

Once information gets into protein, it cannot get out. (Dead end – protein encodes no genetic messages. The protein is put to use but can't be used to synthesize DNA, RNA, or new proteins.)

Genetic information must flow from the nucleus to the cytoplasm because the nucleus holds the DNA while protein construction occurs on ribosomes within the cytoplasm

Transcription results in the formation of a specific RNA molecule.

Transcription requires RNA polymerase. In any given section of DNA, only one of the two

sides is transcribed.

The transcribed strand is called the template strand. The untranscribed strand has no name, but is

sometimes called the complimentary strand to make it feel better about itself.

Genes may be active on either side of the DNA molecule.

mRNA, tRNA, and rRNA are all formed by transcription.

How Much of the DNA Molecule Unwinds? During replication, the DNA molecule unwinds all

the way. During transcription, the only part of the DNA

molecule to unwind is the gene for the necessary protein.

After transcription, the DNA molecule rewinds back into the double helix.

Transcription has three components (in order): (1). Initiation, (2). Elongation (3). Termination

Initiation RNA polymerase binds to a site on the DNA molecule

called a promoter. Each gene has one promoter. RNA polymerase binds to the promoter and starts unwinding the DNA strands at the initiation site. RNA polymerase moves in the 3' to 5' direction along the template strand so that complimentary RNA molecules are synthesized in the correct 5' to 3' order with new

RNA nucleotides attached at the 3' end.

Not all promoters are equally efficient. Those that bind RNA polymerase effectively are transcribed constantly, and those that attach poorly are found at the beginning of genes that are less frequently transcribed.

Elongation Like DNA polymerase, RNA polymerase adds new

nucleotides to the 3' end of the growing strand. As the RNA polymerase moves along the template

strand, unwinding the next stretch of DNA, it rewinds the stretch of DNA that it just processed.

Termination Just like promoters, genes have base sequences that

identify the "end" of the gene. It is at this site that RNA polymerase separation occurs and mRNA synthesis is completed.

This strand of mRNA will undergo further processing before it exits the nucleus and is itself translated in the cytoplasm. More later.

The Genetic Code The genetic information transcribed in mRNA consists of

a number of 3-letter "words". Each sequence of three nucleotides (the "letters") along the chain specifies a particular amino acid. This "three letter word" is called a codon.

•There are 64 different codons (43). •There are 20 different amino acids used in polypeptide

synthesis.

As a result, most amino acids have more than one codon.

Leucine, serine, and arginine are the tri-champions with 6 codons each.

AUG (which codes for methionine) is also the start codon, the initiation signal for translation.

There are three nonsense (or “stop”) codons: UAA, UAG, and UGA

Stop codons are chain terminators. When the polypeptide assembly machinery reaches one of these codons, it stops and polypeptide elongation ceases.

This code is universal and applies to all species on the planet.

Exceptions for the nonsense codons UAA, UAG, and UGA:

These are not the codons that are used in the mitochondria and chloroplasts.

There are several groups of protists wherein the codons UAA and UAG code for glutamine rather than performing as "stop codons". It is not known why.

Scientists "cracked" the Universal Genetic Code in the early 60s.

•Today, biologists can make artificial DNA with known base sequences and "splice" the DNA into bacteria, causing them to synthesize proteins that no organism has ever made before!http://www.youtube.com/watch?v=YM2X1c4K1x0

Prokaryotic mRNAs are translated immediately. •Eukaryotic mRNAs are changed considerably before they

are translated. •Again, translation refers to a change from a nucleotide

sequence code to an amino acid sequence in a polypeptide.

•Translation occurs at the ribosome, which binds to mRNA

http://www.youtube.com/watch?v=D5vH4Q_tAkY

What is needed for Translation? •Well, for sure, a newly synthesized strand of mRNA (from

transcription). •tRNA to carry amino acids to the ribosomes •Ribosomes to bind together amino acids to make protein •Amino acids, the monomers of proteins •Enzymes to regulate every step in this biochemical process.

What is tRNA?

•A tRNA molecule is small nucleic acid molecule consisting of only about 75-80 nucleotides.

•At the 3' end of every tRNA molecule is an "amino acid binding site" to which one amino acid attaches.

•At about the midpoint is a group of three bases, called the anticodon that serves as a contact point for the newly synthesized mRNA molecule from transcription.

Unfolded transfer RNA (left) has a clover-leaf shape. In the cell, it folds into a more compact L shape (right). The sequence of each tRNA molecule differs, but includes an invariant amino acid binding end. The anticodon is unique for each type of amino acid.

•At the contact point (3 bases), mRNA and tRNA are antiparallel.

•There are about 45 different tRNA molecules.* Recall that 61 (64 minus 3 stop codons) different codons

encode the 20 amino acids in proteins. Does this mean that the cell must produce 61 different tRNA molecules, each with a different anticodon? No....the cell gets by with about 45 different RNA molecules because the specificity for the base at the 3' end of the codon is relaxed. This phenomenon, called wobble, allows the alanine codons GCA, GCC, and GCU all to be recognized by the SAME tRNA (to cite one example)

The "job" of tRNA is to transfer amino acids to ribosomes so that they may be used to make protein (this is protein synthesis!).

•Each tRNA molecule is specific in the amino acid that it transports. That is to say that each of the 45 different tRNA molecules will carry one type of amino acid only at

its amino acid attachment site.

What are Ribosomes? As you will recall, a ribosome serves as a

"workbench" for protein assembly Ribosomes are small organelles, but they are

much larger than the molecular components they interact with (mRNA,tRNA).

Each ribosome consists of two subunits. •The large subunit consists of 3 different molecules of

rRNA and about 45 different protein molecules. •The small subunit consists of 1 rRNA molecule and about

33 different protein molecules. When not active in the translation of mRNA, the ribosomes

exist as separated subunits. Ribosomes are non-specific, they may be used as

assembly sites for many different polypeptides.

Initiation: The small ribosomal subunit binds to the leading edge of the

mRNA chain. (The mRNA chain simply diffused through the nuclear envelope into the cytoplasm.)

Remember: the start codon in the genetic code is AUG. This means the first amino acid in the polypeptide will be methionine. (Sometimes this amino acid is removed later by an enzyme. Not all proteins start with methionine).

The anticodon of a methionine-containing tRNA binds to the mRNA codon.

Next, the large subunit joins the complex.

http://www.youtube.com/watch?v=6VV0fKxRcHw

Elongation: During elongation, amino acids are added (one at a time) to

lengthen the amino acid chain. •The first tRNA (bearing methionine) currently lies in the P

site of the ribosome. •The mRNA codon at the A site forms hydrogen bonds with

the anticodon of an incoming molecule of tRNA carrying its specific amino acid.

•This step requires the hydrolysis of two molecules of GTP.

An rRNA molecule of the large ribosomal subunit, acting as a ribozyme, catalyzes the formation of a peptide bond that joins the Methionine to the amino acid borne by the newly-arrived tRNA molecule. NOW, the polypeptide chain consists of 2 amino acids!

The Methionine detaches from its tRNA. The tRNA at the P site (having lost its methionine)

moves to the E site and leaves the ribosome.

The ribosome now translocates (moves) the 2nd tRNA from the A site to the P site.

•A new (3rd) tRNA now comes to occupy the A site, its anticodon matching the codon of the mRNA molecule.

•In this way, amino acids are added to a growing polypeptide chain, one at a time -10 every second!

•A newly arrived tRNA molecule attaches first at the A site, then moves to the P site, and finally to the E site where it exits the ribosome.

http://www.youtube.com/watch?v=yhBxHUBYeRk

http://www.youtube.com/watch?v=v6h5sXfJ72Q

Termination: •Elongation continues until a stop codon in the mRNA

reaches the A site of the ribosome. •These stop codons (UAA UAG UGA) act as signals to

stop translation. •A protein called a release factor binds directly to the

stop codon in the A site. •This release factor causes the addition of a water

molecule to the end of the polypeptide chain. •This reaction hydrolyzes the completed polypeptide

from the tRNA that is still at the P site, freeing the polypeptide from the ribosome.

http://www.youtube.com/watch?v=a-bEwWQHoQQ

http://www.youtube.com/watch?v=nQHkTL7YaJs

Go over questions in notes…..

Polysomes (Polyribosomes) •Several ribosomes can translate a single mRNA

molecule at once, producing multiple copies of a protein at the same time.

•This method yields efficiency and speeds up the rate of protein construction.

•A polysome is like a cafeteria line - the person close to the register has a full tray of food, while the poor soul farther back in the line has one lonely dish of applesauce.

http://www.youtube.com/watch?v=llNwYz9qcB8