|Introduction to Cell & Molecular Biology (BIOL121) - Dr. S.G. Saupe (email@example.com); Biology Department, College of St. Benedict/St. John's University, Collegeville, MN 56321|
More Molecular Genetics: Transcription & Translation
I. How is the Genetic Message Read?
We know that the sequence of nucleotides in DNA is the genetic blueprints. But, how is this nucleotide sequence converted into a phenotype? Some evidence:
A. Inborn Errors of Metabolism
Refers to diseases, caused by a metabolic deficiency, that run in a family. For example, phenylketonuria (PKU) is caused by high concentrations of the amino acid phenylalanine (phe) in the bloodstream. These individuals lack a critical enzyme to convert phe to tyrosine. Excess phe is converted to other products which can cause mental retardation. This can be controlled by limiting the dietary intake of phenylalanine (check a pop can). Since this condition is inherited, it suggests that the failure to make an enzyme (=protein) is specified by inheritance. Archibold Garrod (1909) was one of the first to make this connection when he studied alkaptonuria, a condition in which the urine turns black.
B. Beadle & Tatum
They worked with the bread mold Neurospora and studied mutants that were obtained by exposure to x-rays. Just like the mutant yeast we used in lab that can�t grow in a minimal medium that lacks adenine, the mutant Neurospora couldn't grow in a minimal medium. They would grow if supplemented with the appropriate materials, just like our mutant yeast grows if supplemented with adenine. Nutritional mutants such as these are called auxotrophs.
In particular, they studied the chemical pathway for making arginine, one of the amino acids commonly found in proteins. This pathway required three enzymes (let's call them A, B and C) which converted a starting material (let's call it a precursor) to ornithine to citrulline and finally to arginine, respectively. Thus, each conversion was catalyzed by a different enzyme. We can summarize this reaction:
precursor → (enzyme A) → ornithine → (enzyme B)→ citrulline → (enzyme C) → arginine
Observations and Conclusions:
They knew that only wild type (no mutations) Neurospora grow in minimal medium. They then tried to identify different mutants. If there was a mutation in enzyme A, it couldn�t grow unless the growth medium included ornithine, citrulline or arginine. If the block was in enzyme B, then Neurospora would grow if supplemented with citrulline and arginine. And, if the block was at enzyme C, it would only grow if supplemented by arginine. By performing such experiments, they concluded that genetic information must specify enzymes; or more simply stated: ONE GENE = ONE ENZYME. This was later modified to one gene = one protein, and finally modified to one gene = one polypeptide, since some proteins are made from more than one polypeptide chain.
The phenotype is the result of proteins, many of them enzymes, that are specified by the DNA code.
II. The Central Dogma
This idea explains how DNA (genetic instructions) determines the phenotype. According to the Central Dogma, an idea first offered by F Crick, the genetic instructions are stored as DNA. The message in the DNA is converted to RNA during a process called transcription. The message in the RNA is then used to make a protein during a process called translation. We can diagram this process as follows:
DNA (genotype) → transcription → RNA → translation → protein (phenotype)
Take Home Lessons:
the process of converting the message in DNA into RNA.
Occurs in the nucleus
the process of converting a message in RNA into a protein.
In other words,
'translation' is a short, cool way of saying, �protein synthesis.� Occurs at
the ribosomes in the cytosol
Proteins are the end product of the Central Dogma.
The proteins can be used directly (i.e., hemoglobin, insulin, collagen) or
function as enzymes.
RNA can be an end product of the Central Dogma. In
other words, the RNA that is produced during transcription may, itself, be the
A Goofy Analogy - Barbecued
Let�s use an analogy to help us understand this process - Imagine a reference library where none of the books can circulate. The library building is analogous to the nucleus. The books are analogous to chromosomes - rich with "genetic" information that can never leave the library. Now, let's assume we want to cook up a gourmet meal featuring, say, barbecued cat. But, we need directions. So we go to the library and find the Dead Cat Cookbook. But, we can't take the book back to our kitchen to prepare supper because the book can't leave this library, just as the DNA/chromosomes can�t leave the nucleus. So, what should we do? Of course, we'll make a copy! Transcription is analogous to making a photocopy of a specific section of the book. The photocopied recipe is analogous to the RNA. The ribosome is analogous to the kitchen. Using the recipe in the kitchen to make "barbequed cat" then is analogous to a protein being made at the ribosome from the instructions in RNA via translation.
Now for the delicious and juicy details....
III. RNA - A Quick Primer
RNA - acronym for ribonucleic acid, a type of nucleic acid
RNA is similar to DNA - both are polymers of nucleotides
B. RNA differs from DNA in a few significant ways
RNA is single stranded, not double-stranded
RNA nucleotides have the sugar ribose instead of deoxyribose
RNA is generally shorter
RNA has the nitrogenous base uracil in place of thymine - thus, the four RNA nucleotides are adenine, guanine, cytosine and uracil
C. Types of RNA.
There are three types of RNA:
rRNA - ribosomal RNA - makes up the ribosome, most abundant type
mRNA - messenger RNA - carries the genetic message to the ribosome to be translated into a protein, short-lived
tRNA - transfer RNA - shuttles amino acids to the ribosomes for protein synthesis. Activating enzymes (aminoacyl tRNA synthetases) join the tRNA and amino acids. There are about 45 different kinds of tRNA. All are about 70 nucleotides and have one end that is attached to the amino acid, and the other end has a loop where the anti-codon is found.
Transcription is the production of RNA in the nucleus using a DNA template = RNA synthesis
A. RNA polymerase
This enzyme is responsible for assembling the protein during transcription. In eukaryotes, there are different RNA polymerases (I, II, III) that have slightly different roles during transcription.
RNA polymerase binds to a specific sequence of nucleotides in the DNA at the beginning of gene called the �promoter�. The promoter is essentially a road sign that tells RNA polymerase, "Hey, the gene is over here!" Within the promoter is a specific site on the DNA where RNA synthesis actually begins; this is called the "initiation site." This region is characterized by the nucleotides TATA (TATA box).
RNA polymerase opens up the DNA and then begins to match RNA nucleotides with the DNA nucleotides. This process is similar to replication except that: (a) RNA nucleotides are used (i.e., they have ribose instead of deoxyribose); and (b) uracil substitutes for thymine.
Transcription begins about 20 nucleotides downstream from the TATA box
Approximately 100 nucleotides upstream from the site of the beginning of transcription in the promoter region is an sequence of nucleotides in DNA that facilitate binding of general transcription factors (proteins that are required for the process to work).
Only one strand of the DNA is read into RNA (called the template or sense strand). The other DNA strand simply serves as the complementary strand - it is not used during transcription. One side of the DNA may be the sense strand for one gene, but for another gene, it may be on the other side of the DNA.
RNA is made in the 5� to 3� direction, just like DNA.
The process continues until a termination sequence in the DNA, such as AATAAA, which specifies the end of the gene, is reached. RNA is then released from the DNA, the DNA re-anneals (closes back up), the RNA polymerase looks for another gene to transcribe, and the newly produced RNA goes on its merry way, folding, etc.
C. Fate of the RNA produced during transcription
All three kinds of RNA are produced during transcription: mRNA, tRNA and rRNA. These RNA�s leave the nucleus and go to the cytoplasm where they �get to work�. Thus:
rRNA - links up with proteins to make the subunits of the ribosome.
mRNA - heads out to the cytoplasm to find a ribosome
tRNA - make a beeline to the cytoplasm and becomes activated by linking to the appropriate amino acid. Activating enzymes (aminoacyl tRNA synthetases) join the tRNA and amino acids. There are about 45 different kinds of tRNA. All are about 70 nucleotides and have one end that is attached to the amino acid, and the other end has a loop where the anti-codon is found.
D. mRNA processing
Unlike rRNA and tRNA, the mRNA of eukaryotic cells that is produced during transcription is not a finished product - it must be processed before it can leave the nucleus and be translated into a protein. For this reason it is called primary or pre-mRNA. The mRNA is processed as follows:
the mRNA is capped at the 5' end with a modified nucleotide. The cap serves as a recognition site for later translation. As an analogy, on our photocopied recipe, we might write a special note on the top about where to file our recipe.
a poly A (adenine) tail is added to the 3� end. The tail apparently tells the cell to protect this mRNA from destruction. And, we could put another note at the bottom of the recipe giving our opinion. �Yummy, barbequed cat is mighty tasty - a keeper recipe!�
snip out introns (regions that are transcribed but not translated). Exons are regions that are transcribed and translated. This would be analogous to finding that our cat recipe has randomly scattered paragraphs of gibberish. We would take our scissors, snip these out, and tape the recipe back together. Spliceosomes and ribozymes are the �scissors.�
the final product is "mature mRNA"
IV. Translation or, Protein Synthesis
Using the instructions in mRNA, a protein is created (just like our recipe specifies a tasty treat.)
A. The Genetic Code
The message in mRNA is encoded as a sequence of four nucleotides (A,G,C,U), but a protein has different 20 acids? How can a four letter alphabet be converted to a 20 letter one?
If one nucleotide codes for 1 amino acid, then only four possible amino acids could occur in a protein (4n = 41 = 4). If two nucleotides code for 1 amino acid, then 16 possible amino acids could occur in a protein (4n = 42 = 16). Note: we are still at least four short. If three nucleotides code for 1 amino acid, then 64 possible amino acids could occur in a protein(4n = 43 = 64). This would more than allow for 20 different amino acids, plus could account for punctuation marks. Thus, it makes sense that the genetic code is written in groups of three nucleotides, triplets, called a codon.
A. If you mix some mRNA, an extract of bacteria, some amino acids and other goodies and wait a little while, a protein is formed that has just under 1/3 as many amino acids as nucleotides. Thus, 3 nucleotides/amino acid (triplet code).
B. Some clever experiments by Marshall Nirenberg and others demonstrated the nature of the codons and what they specify (see codon table in text). For example, Nirenberg found that an artificial mRNA comprised of only U produced a peptide with all phenylalanine. Thus, UUU is the codon for phenylalanine.
The code is redundant
Most amino acids are specified by more than one codon. This is the reason that the code is sometimes called �redundant�. Or in other words, there are synonyms for any given amino acid in a protein
The code is NOT Ambiguous. In other words, a
specifies only a single amino acid.
The code contains punctuation
marks. In other words, there are start (AUG) and stop
codons (UGA, UAG, UAA)
The genetic code is universal (with a few exceptions) - which suggests that it evolved very early during the evolution of life.
Made of rRNA and protein. Ribosomes have two subunits, one larger than the other. The larger subunit is comprised of 3 molecules of rRNA and a bunch of proteins (45) while the smaller subunit is made of 1 rRNA and associated proteins (33). The two subunits (halves) of the ribosome don't come together until they start to work (see below). They don't come together to form a functional ribosome unless there is an available piece of mRNA. Ribosomes have two binding sites, called P and A.
C. More Juicy Details
The small ribosome subunit binds to the recognition site (promoter) on the mRNA
the initiation tRNA (which is carrying methionine) binds to the start codon (AUG) on the mRNA (in the P site).
the large ribosome subunit joins the crowd; the ribosome is now complete and functional.
another tRNA carrying the appropriate amino acid (the codon of the DNA matches the anti-codon loop of the RNA) binds to the second site (A).
Adjacent amino acids are linked together by covalent (peptide) bonds; the large subunit of the ribosome catalyzes this reaction. Specifically rRNA acts as a catalyst, called ribozyme, which is obviously an exception to the "enzymes are catalysts rule."
The tRNA that has donated the methionine is released from the mRNA and heads back to the cytoplasm to get charged with another methionine
As the polypeptide chain is built, the mRNA shifts along the ribosome. Each time, the open A site allows another tRNA to match up with the appropriate mRNA codon.
The process continues until a stop codon is reached. This causes the protein, mRNA to detach from ribosome subunits.
D. Protein Processing
As the ribosome constructs the protein, the cell must decide what to do with the finished product. Some proteins remain in the cytoplasm, others are sent to the endoplasmic reticulum. The final destination for a protein is determined in large part by the actual structure of the protein. Specific sequences of amino acids in the protein serve as signals to target the protein to specific organelles.
Once produced, the finished protein may be further modified by: (a) removing amino acid fragments (proteolysis); (b) adding a sugar(s) (glycosylation); or (c) adding a phosphate (phosphorylation). Collectively these modifications are necessary for the proper functioning of the protein.
Back to supper
Translation is analogous to actually cooking a cat from a recipe. The ribosome is analogous to the kitchen, the site where the product is prepared. The butter, flour, spices, etc. are analogous to the amino acids. You, the cook, are like the tRNA bringing the ingredients to the stove/mixing bowl. And the final dish, is analogous to the protein. Continuing with this analogy, replication would occur when the entire library, is copied. A copy would be distributed to a new library built to house this collection.
Last updated: July 14, 2009 � Copyright by SG Saupe