Molecular Genetics

Semiconservative Replication

I. The difference between one organism and another organism, at its most basic level, is a difference between the biochemical reactions that the two different organisms can perform. As mentioned earlier, every biochemical reaction that occurs in or around a cell requires the activity of a specific enzyme. Virtually all enzymes are proteins. The difference between one protein and another protein stems from differences in the sequence of amino acids that make up the proteins. It is very important when making a protein that the amino acids are added in the proper order. How this is accomplished involves using a molecule of ribonucleic acid (RNA) as a template while the protein is synthesized. Synthesis of the RNA molecule requires using deoxyribonucleic acid (DNA) as a template. Every protein that a cell can make must be coded for in that cell's DNA. In other words, everything an organism is and is capable of is determined by its DNA.

A. In DNA, the sequence of bases in the long nucleotide polymer is where all of the "information" is stored. How this sequence of nucleotides is used to synthesize a protein with a specific sequence of amino acids will be discussed shortly.

B. The sequence of nucleotides in the DNA that codes for a particular protein and those parts of the DNA that control when the DNA is used to make that protein are referred to as the  gene for that protein.  

1. The human genome is estimated to have 3.2 billion nucleotides.  This DNA encodes 26,000-35,000 genes (each gene holds the information for making a specific protein).
2. Bacterial DNA is far less complex than human DNA. It has fewer genes and far less complex systems of gene regulation. Bacteria make far fewer proteins than the average eukaryotic organism.

C. The bacterial chromosome is a long, circular piece of double stranded DNA that contains most of the genes of the bacteria. It is highly complexed with proteins that are attracted to the DNA. The chromosome is not in a separate compartment and thus takes up a large percentage of the cytoplasmic volume of the bacterial cell. The genetic material of eukaryotic cells also consists of DNA complexed with proteins but the eukaryotic DNA is not circular.  In the eukaryotic organism the proteins that associate with the DNA are referred to as histones. Humans have 46 chromosomes.

II. Before any cell (either eukaryotic or prokaryotic) can divide, the genetic information stored in DNA must be copied with very few mistakes. This requires a mechanism that will produce two identical double helices from one initial double helix. The process by which the DNA sequence is faithfully duplicated is known as semiconservative replication.

A. Replication begins with the association of specific proteins known as helicases that cause the DNA helix to unwind. As the helicase unwinds the  DNA supercoils are introduced into the DNA.  These supercoils must be removed so that the DNA can continue to be unwound.  The action of an enzyme known as DNA gyrase removes the supercoils and allows unwinding of the DNA to proceed.   

B. Once unwound a short piece of ribonucleic acid which is complementary to a region of the DNA is made by an enzyme known as primase ( primase is one of several RNA polymerases). This short piece of RNA is known as a primer. Without the primer the rest of DNA replication can not occur. This is a very important idea and will be returned to when we discuss the polymerase chain reaction (PCR) method of DNA analysis.

C. Immediately, an enzyme known as DNA polymerase III and several other protein molecules, attach to the DNA strand and begin synthesizing a new strand of DNA. A second DNA synthesizing enzyme, DNA polymerase I, will remove the RNA primer and replace it with deoxyribonucleotides.

D. DNA is merely a polymer of deoxyribonucleotides covalently bound together. The covalent bond forms between an oxygen attached to the 3' carbon of one nucleotide and the phosphate group attached to the 5' carbon of another nucleotide. The nucleotides are added one at a time.

E. The addition of nucleotides into the new strand of DNA is not random. Addition of nucleotides is guided by complementary base pairing between the incoming nucleotides and the nucleotides in the old strand of DNA. For this reason the old strand is referred to as a template. The polymerase only adds a base to the new strand of DNA if it correctly base pairs with the base in the template strand (A-T and G-C). This specific interaction between nucleotides is known as complementary base pairing.

F. When replication is complete two double helices will be formed. At this point a cell can undergo division and give each new cell a full set of DNA.
 

Transcription and Translation

I. Ribonucleic acid (RNA) is essential in the utilization of the information stored in the DNA for the synthesis of proteins.

A. Ribonucleic acid is a polymer of ribonucleotides and is similar to a single strand of DNA. The backbone of RNA consists of ribonucleotides that contain the sugar ribose (as opposed to the deoxyribose found in the backbone of DNA) and a phosphate group. The ribonucleotides are covalent bound together. As in DNA, a covalent bond is formed between an oxygen attached to the 3' carbon of one ribonucleotide and the phosphate group attached to the 5' carbon of the next nucleotide.

1. The difference between ribose and deoxyribose is that there is an -OH group in place of a -H attached to one of the carbons of the sugar. This seemingly minor difference keeps ribonucleotides from being used by DNA polymerase (in the synthesis of DNA) and deoxyribonucleotides from being used by RNA polymerase (in the synthesis of RNA).
2. Another difference is that in RNA, the nitrogenous base thymine is not present but is replaced by uracil.

B. RNA usually exists as a single strand though often the molecule will complementary bind with itself thus folding itself into distinct shapes. These shapes are necessary for the proper function of certain RNA molecules.

II. Classes of RNA Several classes of RNA exist. Though structurally they are identical, both the functions they serve and enzymes which produce them are different.

A. Messenger RNA (mRNA) serves as a template for protein synthesis. This allows amino acids to be put together in a specific order. The sequence of nucleotides in the RNA molecule determines the sequence of amino acids in proteins. The sequence of amino acids determines what the protein can do.

B. Transfer RNA (tRNA) is attached to specific amino acids and guides them to the protein synthesizing complex where it will direct their addition to the growing protein polymer. We will not be discussing the mechanism by which specifc tRNA molecules are properly linked with the appropriate amino acids.

C. Ribosomal RNA (rRNA) complexes with several proteins to form a ribosome. Ribosomal structure and function will be discussed momentarily.
 

III. All classes of RNA are synthesized in a process known as transcription, which is similar to DNA synthesis but utilizes different enzymes. The DNA is unwound in a specific region and enzymes known as RNA polymerases covalently bind ribonucleotides together as the ribonucleotides complementary base pair with the nucleotides in the DNA. Thus the base sequence of the DNA serves as a template for the synthesis of a strand of RNA. The resultant RNA molecule is complementary to the DNA.

A. One major and important difference between RNA and DNA synthesis is that RNA transcription uses only a limited and specific portion of the one of  DNA strands. (DNA replication involves synthesizing two complete complementary strands of DNA.)

B. Proteins known as promoters or transcriptional factors bind to the DNA in such a way as to allow only a specific region of the DNA to be used. These proteins bind to a specific sequence of nucleotides known as the promoter region of the DNA. They determine where RNA polymerase begins transcribing the messenger RNA.  Promoters specifically increase the transcription of one particular gene. Binding of a promoter protein to the promoter region of the DNA specifically "turns-on" transcription of the DNA sequence "down stream" from the binding site. Consequently, a RNA molecule is produced that is complementary to the DNA sequence of that region.  The binding of the promoter to the promoter region and the initiation of the transcription process is often referred to as "turning on a gene". 

C. Another type of protein that effects which regions of the DNA get used in the transcription of RNA are proteins known as repressors. A repressor binds to the DNA and inhibits the activity of RNA polymerase. In other words they "turn off" a gene.

D. A major difference between the mRNA in the prokaryoticcell and that made in the eucaryotic cell is how the mRNA molecule is handled after transcription is complete but before a mRNA leaves the nucleus. The RNA molecule is altered so that it will be recognized by the eucaryotic ribosome in a group of reactions collectively termed as post-transcriptional modifications. A long series of adenine nucleotides known as the poly-A tail are added to the 3'-end of the mRNA. A "cap structure" is added to the 5'-end. This cap is a nucleotide added in the opposite orientation. Intervening sequences or introns are cut out of the mRNA and the remaining exons are spliced together. The mRNA then leaves the nucleus and enters the cytoplasm. Without these modifications the mRNA could not be used as a guide for protein synthesis. These modifications do not occur in the prokaryotic cell. 
 

IV. As mentioned earlier, proteins consist of polymers of amino acids covalently bound together. The covalent bond between amino acids is known as a peptide bond. Proteins are synthesized in a process known as translation. Translation requires ribosomes, a template of mRNA and many tRNA molecules specifically linked to the various amino acids (amino acyl-tRNA).

A. Translation begins with the binding of a mRNA to the small subunit of a structure known as a ribosome. A specific amino acyl-tRNA then associates with the mRNA. The large subunit of the ribosome then attaches and the complex (known as the initiation complex) moves down the mRNA molecule scanning for a specific three- nucleotide base sequence (A-U-G) known as the start codon. When this sequence is encountered, the tRNA base pairs with the sequence and waits for another tRNA to attach to the next three bases in the mRNA.

B. The anti-codon region of the each tRNA is complementary to the sequence of three nucleotides of the mRNA to which it will associate by complementary base pairing. The tRNA, carrying a specific amino acid, will associate with the mRNA by way of the base pairing between the nucleotides of the anti-codon and those of the mRNA. At this point the ribosome will covalently link the amino acid carried by the tRNA to the growing amino acid polymer. Thus, amino acids are specifically added one at a time as the ribosome moves down the mRNA.

C. The mRNA is recognized by the tRNA three bases at a time. These three base sequences are known as codons. There are 64 different codons. Most amino acids are coded for by more than one codon. Several codons exist that have no complementary tRNA (and thus code for no amino acid). These are the nonsense or stop codons. When one of these is reached the translational complex can go no further. It quickly falls apart and the protein is released.
 

V. As mentioned above, the mRNA molecule will be translated one codon (three nucleotides) at a time. Each of the codons codes for a specific amino acid. This is referred to as the genetic code. The genetic code is virtually the same for all living organisms. So a gene taken from a human cell, introduced into a bacteria cell, can be used by that bacteria cell to make the same protein that the human cell would have made. Refer to your textbook or teh following website for a copy of this code.  http://www.cbs.umn.edu/~amundsen/chlamy/code.html

A. It should be noted that even though the same genetic code is used by both bacterial cells and eucaryotic cells the ribosomes that translate the mRNA have some differences. Certain antibiotics (chloramphenicol, erythromycin, tetracycline, etc.) adversely effect prokaryoticribosomes to a much greater degree than they do eukaryotic ribosomes.

B. Certain proteins that are made in eukaryotic cells are processed in the rough endoplasmic reticulum and golgi apparatus. Prokaryotic cells lack these organelles and thus can not affect these modifications. For this reason fungal cells such as yeast are often genetically engineered to produce proteins needed in human therapy.
 

VI. Some genes are always expressed at the same rate. This is referred to as constitutive expression. But many genes are only expressed as they are needed. Controlling the expression of a gene can involve mechanisms that increase the rate at which RNA polymerase begins transcription of a region of DNA (induction), mechanisms that decrease the rate at which RNA polymerase begins transcription of a region of DNA (repression) or a mechanism that stops transcription before it is completed (attenuation). We will examine two sets of genes, the Lac operon and the arg operon.

VII. The Lac operon is a bacterial genetic system that regulates the synthesis of three proteins: galactoside permease, galactose transacetylase and beta-galactosidase. These proteins are enzymes and cytoplasmic membrane transporters that allow the bacteria to import the sugar lactose and break it down to be used for energy. This system of genetic control was worked out by two researchers F. Jacob and J. Monad and was the first genetic regulatory system to be figured out.

A. Generally, organisms do not want to make enzymes for substrates that are not present in their environment. Furthermore, sugars such as lactose are "second choices" for bacteria. If their first choice, glucose, is present they will use it preferentially. So the bacteria need a way to control the proteins that are used to break down second choice sugars. These genes can be transcribed under certain conditions (presence of lactose and absence of glucose) but can be shut off (repressed) under other conditions (high glucose or no glucose but no lactose).

1. The three genes needed by the cell for the use lactose are located next to one another in the bacteria DNA.
2. Only in an environment that has lactose and lacks glucose are the genes for lactose utilization turned on. In the presence of glucose and lactose these genes are not turned on neither are they activated if lactose is absent regardless of the amount of glucose in the environment.
3. To properly understand this system, the reactions to the various environmental conditions described above must be explained.

B. Jacob and Monad successfully explained the inducing role played by lactose. Their explanation involves the activity of a protein referred to as the repressor protein. [This is more properly referred to as the I protein.] The repressor protein is constitutively expressed so it is present in the cytoplasm in constant amounts. In the absence of lactose, this protein binds to the DNA near to the start of those genes needed for the breakdown of lactose. When it binds to the DNA, it blocks the site where transcription should start and so inhibits (or represses) the transcription of the genes.

1. The site in the DNA to which the repressor protein binds is referred to as the operator site (O site) and is a specific sequence of nucleotides.
2. When lactose is present, the lactose combines with the repressor protein and changes its shape so that it can not bind to the DNA at the O site. Thus transcription of these genes is no longer blocked.

C. Why the presence of glucose inhibits the synthesis of lactose metabolizing genes took several years to figure out. Several pieces of information needed to be uncovered first.

1. When glucose is lacking, the cell produces a substance known as cyclic AMP (cAMP). As glucose becomes scarce, the concentration of cytoplasmic cAMP increases.
2. Within the bacterial cell another protein which is constitutively expressed is the cAMP receptor protein (CRP). This protein binds with cAMP and then (and only then) is capable of binding to the DNA.
 

D. The site that cAMP-CRP binds to the DNA is a region next to the genes for lactose metabolism. The binding of the cAMP-CRP protein aids the RNA polymerase in starting transcription of the genes needed to utilize lactose .

E. Now for each environmental condition look at the state that the Repressor is in and the state the CRP protein is in:

1. When lactose is not present, the repressor protein is bound to the region next to the genes for lactose metabolism inhibiting the transcription of these genes. So regardless of the amount of glucose present there will be no transcription of the Lactose metabolizing proteins.
2. When lactose is present but glucose is also present, the repressor protein binds lactose and does not bind to the DNA. But because glucose is plentiful the cAMP is low and no cAMP-CRP complex forms. Without this complex, transcription can not be started.
3. In the case where lactose is present and glucose is absent. The repressor protein binds with lactose and does not bind to the DNA. Furthermore, when the cAMP level is high this results in the formation of cAMP-CRP complexes that bind to the DNA promoting transcription. The genes for metabolism of lactose are transcribed and translated. Thus the cell "turns on" those genes only when they are needed.
 

Link to the original paper published by Watson and Crick regarding the structure of DNA (1953)  http://biocrs.biomed.brown.edu/Books/Chapters/Ch%208/DH-Paper.html

For a paper that changed the world it is remarkably short!!!

MIT Biology   DNA Replication  http://web.mit.edu/esgbio/www/dogma/dogmadir.html

This is a good overview of replication.  It is not specific for prokaryotic organisms.

MIT Biology    Prokaryotic Genetics     http://web.mit.edu/esgbio/www/pge/pgedir.html

Grapes of Staph   Transcription and Translation 

  http://www.cat.cc.md.us/courses/bio141/lecguide/unit4/u4ib5.html

The Human Genome Program  http://www.ornl.gov/hgmis/

This is the Department of Energy's official site. Your tax dollars doing some good work!!