Molecular Genetics

I.
Nucleic Acid Structure
A.
DNA.
1.
DNA is a double polymer of nucleotides (ie, it is a double polynucleotide.)
2.
Each nucleotide is composed of
a.
A pentose sugar (in DNA, deoxyribose), with
b.
A phosphate functional group attached to its 5' carbon and
c.
A nitrogenous base attached to its 1' carbon.
3.
There are four kinds of nitrogenous base in DNA, which can be divided into two basic types.
a.
Purines (Adenine and Guanine) have a double ring structure.
b.
Pyrimidines (Thymine and Cytosine) have a single ring structure.
4.
Nucleotides are linked together by bonding the phosphate of one nucleotide to the 3' carbon of another nucleotide.
5.
Repeating this over and over creates a long chain of nucleotides: a polynucleotide. A polynucleotide has "direction"--the 5' and 3' dimensions of all of the nucleotides in the chain will be positioned in the same way. This means that each polynucleotide has a 5' end and a 3' end.
6.
The spine of a polynucleotide is composed of the alternating sugars and phosphates, and is referred to as the sugar/phosphate backbone.
7.
The bases attached to the nucleotides are not part of this structural connection and thus can come in any order. This is the basis of the information-carrying capacity of nucleic acids.
8.
Each DNA is composed of two polynucleotides associated with each other in an antiparallel arrangement. This means the molecules "lie" side by side, one in the 5' to 3' direction, the other in the 3' to 5' direction.
9.
The two polynucleotide strands are held together by hydrogen bonding between facing pairs of bases, one on each strand. This creates a ladder-like appearance. Each "rung" of the ladder is composed of one base pair.
10.
The association between bases in base pairs is precise and exacting. Adenine will accept only Thymine as a partner; Thymine will accept only Adenine. Guanine pairs only with Cytosine; Cytosine with Guanine. This precision partnering is called Complementary Base Pairing, and is the feature of nucleic acid structure which is responsible for all functions performed by DNA and RNA. Adenine and Thymine are complements of each other; Cytosine and Guanine are complements of each other.
11.
The shapes and angles of the bonds involved in the construction of polynucleotides and the face to face pairing of bases creates a twist in the three dimensional double stranded molecule. Thus, whenever mucleic acids are double stranded, they twist into the characteristic double helix formation.
B.
RNA is different from DNA:
1.
The sugar in RNA is ribose. Ribose has one more oxygen than deoxyribose, but is otherwise identical.
2.
The four bases in RNA are Adenine, Guanine, Cytosine and Uracil. Uracil behaves just like Thymine. Its structure is the same, except that it lacks a methyl group on the #5 carbon. Uracil, like Thymine, is complementary to Adenine.
3.
RNA is a single polynucleotide strand rather than a double strand. In many RNAs internal complementarity causes parts of the molecule to base pair with other parts, but there is only one continuous sugar/phosphate backbone.
4.
RNA is much shorter than DNA. A very large RNA may be a couple of thousand bases long; many are only about 80 bases long. DNA is characteristically millions of base pairs long.

II.
DNA Replication
A.
DNA replication is semiconservative. When a DNA molecule creates a copy of itself, what actually happens is that the two strands of the original double helix separate ("unzip") and each acquires a new partnering strand. Thus each new molecule is actually only half new; the other half is one half of the old molecule. The new strands are formed out of free nucleotides which are a significant component of the nucleoplasm in any cell. Nucleotides in the new strand are selected by complementary base pairing to the bases in the old, template strand.
B.
The site of replication is called a replication fork.
1.
The two sides of the molecule are separated for a short distance. Since DNA is most stable (and least vulnerable to damage) in its double stranded configuration, as little of it as possible will be single stranded at once.
2.
Replication proceeds along both sides of the fork.
3.
The primary enzyme responsible for DNA replication is DNA polymerase. It is a very specialized enzyme.
a.
DNA polymerase only performs one task: following the complementary base pairing rules, it adds new free nucleotides to an already growing strand of nucleotides in the new strand of a replicating DNA molecule. This means that DNA polymerase is unable to actually begin the task of replication, since it has no growing strand to which to attach new nucleotides. For this reason, and enzyme called primase (an RNA polymerase), which is not so picky, actually begins the replication process by building a short piece of RNA called a primer. The DNA polymerase can then use the primer as its growing strand and proceed to build the new DNA. The primer is later removed and replaced by DNA.
b.
DNA polymerase is only capable of building a new strand from its 5' end toward its 3' end. This presents a problem, because the two sides of the old DNA are antiparallel. One side of the new strand (called the leading strand) can be directly and continuously constructed from its 5' end to its 3' end. The other strand (the lagging strand) must be constructed in short segments, built backward (ie, built in the opposite direction of the direction the replication fork is moving). These short strands of new DNA are called Okasaki fragments after the man who discovered them. The Okasaki fragments will eventually be connected together to form a continuous strand by an enzyme called DNA ligase. DNA ligase specializes in healing single stranded nicks in DNA. It adds no new material; it simply seals the bond between one nucleotide and its neighboring nucleotide. Note that each Okasaki fragment requires the formation of a primer, which will later have to be replaced.
C.
DNA in eukaryotes is extremely long. It would take a very long time to replicate the whole molecule from end to end via a single replication fork. Each of these long molecules has many sites called Origins. When replication begins, each of these origins becomes a site where the two sides of the DNA "melt" and replication begins. This forms a configuration called a replication bubble. Each replication bubble actually consists of two replication forks, one at each end of the bubble, and traveling in opposite directions. Each replication fork is as described above. The forks progress along the molecule until they meet, thus replicating all of the very long molecule in a number of shorter segments. These segments are ultimately joined together by DNA ligase.
D.
The helical structure of DNA is an intrinsic aspect of its double stranded structure. DNA cannot be double stranded and not twist. Unless there is a mechanism to relax the twists between replication bubbles, the helical turns would compress between the progressing replication forks and create increasing stress on the backbones of the two polynucleotides, eventually resulting in random breakage and damage. This problem is solved by the creation of single stranded breaks (swivels) between origin sites. These nicks are made by an enzyme called DNA topoisomerase (previously "unwindase," then "swivelase"). After the replication bubbles meet, the single stranded breaks are healed by DNA ligase.

III.
Transcription
A.
Basically, transcription involves locally separating the two strands of the DNA of a segment of DNA and asymmetrically constructing an RNA (the transcript) complementary to one side of the DNA (the template or sense strand). The other side of the DNA (the antisense strand) is not involved. When transcription is completed, the DNA molecule is restored to its original state.
B.
There are three kinds of RNA. All are made by the same kind of transcription process.
1.
Messenger RNA (mRNA) is transcribed from structural genes, and carries a copy of the instructions for constructing a protein (which is what a structural gene is).
2.
Ribosomal RNA (rRNA) is transcribed from rDNA, and is part of the structure of ribosomes.
3.
Transfer RNA (tRNA) is trsnascribed from tDNA and performs an important role in translation.
C.
The enzyme responsible for transcription is RNA polymerase. There are three kinds of RNA polymerase.
1.
RNA polymerase I is responsible for transcribing most kinds of rRNA.
2.
RNA polymerase II is responsible for transcribing mRNA.
3.
RNA polymerase III is responsible for transcribing tRNA and the smallest of the rRNAs. tRNA is the smallest kind of RNA, and this polymerase seems to specialize in transcription of small RNAs.
D.
Transcription occurs in three steps.
1.
Initiation
a.
RNA is always constructed from the 5' end to the 3' end of the new molecule.
b.
RNA polymerase must bind to the DNA in a region called the Promoter. This is positioned about 25 bases in front (upstream) of the beginning of transcription.
c.
Each kind of polymerase has its own recognition site. For RNA polymerase II, its promoter site begins with a sequence called the TATA box. The complete sequence of the TATA box is TATAAAA. (In some cases the fifth and seventh bases may be T instead of A.)
d.
In binding, the RNA polymerase causes the melting of a short (about 10 base pairs long) segment of the DNA.
2.
Elongation
a.
The RNA polymerase proceeds along the DNA, constructing an RNA complementary to the template strand of the DNA by bonding nucleotides one at a time, selecting the nucleotides according to complementary base pairing.
b.
The transicription bubble in the DNA remains about 10 base pairs long. The DNA separates and recloses as the polymerase proceeds along. Thus the RNA actually becomes separated from the DNA almost as soon as it is made.
3.
Termination: there is a termination sequence at the end of each gene. When the polymerase reaches this sequence, it releases the new RNA and falls off the DNA. The DNA returns to its normal double stranded configuration. The DNA is ultimately unaltered by being transcribed.

IV.
Translation
A.
mRNA contains a set of instructions for constructing a particular protein.
1.
A protein is a linear chain of amino acids.
2.
The instructions in mRNA specify which amino acids are utilized in the protein and in which order they are to be connected.
B.
The instructions in mRNA are coded. Each "word" in the code is called a codon. The code is:
1.
Triplet (each codon is three bases long),
2.
Redundant (Degenerate) (the 64 possible codons code for only 20 different amino acids, so the code contains a lot of synonyms).
3.
Universal (all known organisms use essentially the same code).
C.
The agent of translation is the ribosome.
1.
Each cell contains millions of ribosomes. As far as we know, any ribosome can translate any message.
2.
A ribosome is composed of two subunits, called the small subunit and the large subunit. Inactive ribosomes are disassembled; the subunits are separate.
3.
In eukaryotic cells, the two subunits between them contain five kinds of rRNA and about 80 kinds of protein. Prokaryotic ribosomes contain three kinds of rRNA and about 50 kinds of protein. (The larger ribosome is often referred to as an 80s ribosome, the smaller prokaryotic ribosome as a 70s ribosome. These are sedimentation values, and refer to the fact that the larger, heavier eukaryotic ribosome sediments faster than the smaller prokaryotic ribosome does.)
D.
The key element in translation is transfer RNA.
1.
A cell produces 61 kinds of tRNA.
2.
Each tRNA is about 80 bases long, with a lot of internal complementarity. This causes a lot of internal base pairing, bending the molecule into a sort of folded cloverleaf shape.
3.
A tRNA has two significant regions.
a.
Each tRNA has one end which is its aminoacyl attachment site. A special set of cellular enzymes (aminoacyl-tRNA synthetases) is responsible for charging the aminoacyl attachment sites of tRNAs with amino acids--one per tRNA.
b.
At the outer edge of one of the loops of the cloverleaf shape is a three-base-long sequence called the anticodon. Saying that there are 61 different kinds of tRNA actually means that there are 61 different anticodons.
4.
When a tRNA is charged with an amino acid, which amino acid it accepts depends upon its anticodon. A specific anticodon is always associated with the same amino acid. For instance, if a tRNA has the anticodon AAA, it will always carry the amino acid phenylalanine--it will never be charged with any other amino acid. The aminoacyl-tRNA synthetases in the cytoplasm of the cell must be very specific and accurate about this, or every protein in the cell will be made incorrectly. This is the reason the genetic code has been so very highly conserved throughout the vast diversification of life via evolution.
5.
Because there are a lot more anticodons than amino acid types, a particular amino acid may be associated with more than one kind of anticodon. For example, the AAA anticodon carries phenylalanine, but so does the AAG anticodon. Thus the redundancy of the genetic code.
E.
The message in the mRNA is colinear with the sequence of amino acids which is to be constructed.
1.
The first codon in the message is always AUG. This codon doubles as the start codon and the code for the amino acid methionine. Thus, every protein when newly made begins with this amino acid. AUG may occur inside a message, in which case it functions simply as the code for methionine.
2.
A typical message is about 150 codons long, though they can vary from less than 100 to several hundred bases long.
3.
The message ends with one of three stop codons. No amino acid is inserted at these positions. There are no tRNAs made with anticodons complementary to these stop codons (thus the fact that there are only 61 anticodons made, rather than the mathematically possible 64).
F.
Translation occurs in three steps.
1.
Initiation involves the formation of an initiation complex which includes the two subunits of the ribosome, the mRNA, the charged AUG tRNA and assorted initiation factors.
a.
The small subunit begins at the 5' end of the mRNA.
b.
It travels along the mRNA until it encounters the first AUG codon.
c.
The tRNA carrying methionine attaches via complementary base pairing between the AUG codon and the anticodon of the tRNA (thus, the met-tRNA has the anticodon UAC).
d.
The large subunit attaches.
e.
A variety of initiation factors is involved; the exact number and identity depends upon whether the cells is prokaryotic or eukaryotic.
f.
GTP is the energy source utilized for translation initiation.
2.
Elongation gradually builds the amino acid sequence of the protein, one amino acid at a time.
a.
A ribosome is only big enough to include two codons of the mRNA at a time. The ribosome is described as having two codon positions, called the A site (aminoacyl site) and the P site (peptidyl site). There can thus be a maximum of two tRNAs within the ribosome at any one time. Because there are only a maximum of nine hydrogen bonds between a codon and an anticodon, their connection is very tenuous, and is unstable unless the ribosome is in position to keep them together.
b.
In the initiation complex, the met-tRNA is occupying the P site.
c.
A second charged tRNA is positioned in the A site according to complementary base pairing between its anticodon and the second codon of the message.
d.
A peptide bond is formed between the methionine and the second amino acid carried by the second tRNA. This bond is formed by the action of the enzyme peptidyl transferase, which is one of the approximately 80 proteins making up the ribosome.
e.
The bond attaching the methionine (aa#1) to its tRNA is broken. At this point, the second tRNA is holding a "chain" of two amino acids, and is still base paired to the second codon in the message.
f.
The ribosome indexes one codon downstream. It is now covering codon #2 and Codon #3, and codon #1 (AUG) is uncovered. tRNA #2 (carrying the 2-amino acid fragment) is now in the P site.
g.
No longer stabilized by the presence of the ribosome, the connection between the AUG and its anticodon breaks; the tRNA floats off, gets grabbed by the appropriate aminoacyl-tRNA synthetase, and gets recharged with a fresh methionine amino acid. tRNAs are used over and over again.
h. **
The tRNA whose anticodon is complementary to codon #3 is positioned in the A site, along with its amino acid (aa#3).
i.
Peptidyl transferase bonds AA#2 and AA#3; the connection between tRNA #2 and aa#2 is broken. tRNA #3 (sitting in the A site) now has a chain of three amino acids attached to it.
j. ***
The ribosome indexes over to cover codon #4; codon #2 is uncovered and loses its tRNA, which goes off to get recharged.
k.
The tRNA whose anticodon is complementary to codon #4 is positioned in the A site, along with its passenger, AA#4.
l.
Etc., etc., etc. The sequence from ** to *** is repeated until the ribosome indexes to position a stop codon in the A site.
3.
Termination occurs when the ribosome has fininshed manufacturing the protein.
a.
The termination signal is apparently reaching a point where there is no tRNA which has an anticodon complementary to the codon in the A site.
b.
When this point is reached, the ribosome releases the now completed protein, which will then be modified and processed before assuming its task in keeping the cell running properly.
c.
The final tRNA is released to be recharged and reused,
d.
The two subunits of the ribosome fall off the mRNA.
G.
Most mRNAs will be translated more than once. We don't know everything about just what mechanism is used to control how many times a particular mRNA is translated, though it may have something to do with a typical post-transcriptional modification to which most mRNAs are subjected--the attachment of a poly-A tail.
H.
A number of ribosomes will often translate the same message at the same time. They function sequentially; the first will begin, index along a ways, then a second will being, and when it has moved far enough, a third, etc. In electron micrographs of the cytoplasm of cells, these configurations can be seen as a "conga line" of ribosomes. The mRNA is too thin to see, but the ribosomes are visible and obviously chained. This configuration is called a polyribosome or polysome.
I.
Ribosomes may function many, many times. there is no reason to suppose that the same small and large subunits are reunited the next time a ribosome functions.
J.
tRNAs may be used over and over. Because of the nature of the role the tRNA performs in translation, it may be used in the translation of any message.