1. Protein Synthesis -The “Stuff of Life”
Graphic
The pink cell is a T cell and the blue cell is an antigen presenting cell, more specifically a dendritic cell.
Protein Synthesis -The “Stuff of Life”

2. Proteins Proteins are the “workhorse” molecule found in organisms.
The blue print for proteins is coded in the DNA of the organism.
DNA contains genes that determine the phenotype of an organism or "what we look like". DNA codes for the synthesis of proteins. Proteins are responsible for the phenotype.
Humans can make over 200,000 proteins actually much more than that if the immune system is taken into consideration.
Made of polypeptide chains. Proteins have primary, secondary, tertiary and quaternary structure.
There are 20 different amino acids and the average polypeptide chain is 400 amino acids long.
The part of the DNA that codes for a particular polypeptide chain is known as a gene. 2 2

3. Uses of Proteins Uses of proteins Enzymes (catalase)
Structure (silk, hair, nails)
Antibodies
Movement(muscle, flagella)
Hormones (insulin)
Carry gases (hemoglobin)
Storage of amino acids (albumin)

4. History
In 1909, British physician Archibald Gerrod first suggested that genes dictate phenotypes through enzymes that catalyze specific chemical reactions.
He thought symptoms of an inherited disease reflect an inability to synthesize a certain enzyme.
Linking genes to enzymes required understanding that cells synthesize and degrade molecules in a series of steps, a metabolic pathway.
Archibald Garrod was the first to connect a human disorder with Mendel's laws of inheritance. He also proposed the idea that diseases came about through a metabolic route leading to the molecular basis of inheritance.
Garrod was studying the human disorder alkaptonuria. He collected family history information (as well as urine) from his patients. Based on discussions with Mendel advocate William Bateson, Garrod deduced that alkaptonuria is a recessive disorder. In 1902, Garrod published a book called The Incidence of Alkaptonuria: a Study in Chemical Individuality. This is the first published account of a case of recessive inheritance in humans.
1909 Archibald Gerrod suggested that genes determine phenotype through defective enzymes controlling biochemical pathways.

5. Work of Beadle and Tatum
Beadle and Tatum's key experiments involved exposing the bread mold ,Neurospora crassa to x-rays, causing mutations. In a series of experiments, they showed that these mutations caused changes in specific enzymes involved in metabolic pathways. These experiments led them to propose a direct link between genes and enzymatic reactions, known as the "one gene, one enzyme" hypothesis. They received the Nobel Prize for Physiology or Medicine in 1958 for their research.
George Beadle and Edward Tatum established the link between genes and enzymes in studying bread mold, Neurospora crassa.

6. Conclusion One gene produces one enzyme. Later it was modified
One gene produces one protein.
One gene produces one polypeptide chain.
Today the definition for a gene is a sequence of DNA molecules that can direct the synthesis of some sort of molecule product. i.e. genes do not all code for a protein, but all do code for an RNA molecule. Some of those RNAs are translated into protein, but many serve other functions, such as gene regulation.
The molecules can be a polypeptide chain, protein, or RNA. There are genes that make pieces of RNA that do not produce a protein product. For example tRNAs are coded for by a DNA gene but yet it does not make a polypeptide chain.

7. Overview of Protein Synthesis
DNA is located in the nucleus
Proteins are made in the cytoplasm
RNA is the intermediate between the DNA code and the actual synthesis of a protein
The DNA that codes for the proteins is located in the nucleus but proteins are actually made in the cytoplasm. There must be an intermediate that can take the code (instructions) out to the cytoplasm so that the protein can be made.
RNA is the intermediate that takes the code out to the ribosome so that the protein can be made.
Protein synthesis has two major parts.
Transcription-DNA nucleotides found in a gene are used as a template to make a molecule of RNA
Translation- RNA template or mRNA is used in conjunction with ribosomes, tRNA with attached amino acids to produce a polypeptide chain.

8. Structure of RNA versus DNA
RNA vs. DNA
RNA DNA
Single stranded Double stranded
Ribose Deoxyribose
U instead T T instead of U
Nucleus and cytoplasm Restricted to nucleus & organelles
Multiple uses Used as template for RNA synthesis and proteins

9. Three Main Types of RNA There are different types of RNA
mRNA-carries the information from the DNA gene to the cytoplasm. Determines the sequence of amino acids for a protein
tRNA-brings the correct amino acid to the ribosome and mRNA in translation
rRNA-found on ribosomes and used to "connect" the tRNA to the mRNA
snRNA-found on spliceosomes. Used to remove introns.
SRP RNA-part of the signal recognition particle used to bring a translating ribosome to the E.R. and threads the emerging polypeptide chain into the lumen of the E.R.

10. Genetic Code
Amino acids are coded for by a triplet of DNA nucleotides called a codon.
Notes on codons-
1. There 64 codons- 61 code for amino acids. There is "redundancy" in the code; more than one codon codes for the same amino acid.
2. Three codons code for stop.
3. One codes for start and also for methionine.
Since DNA code is transcribed into mRNA, the genetic code in books is described in terms of mRNA codons.

11. Genetic Code
Marshall Nirenberg and Heinrich Matthaei determined the first codon for an amino acid. It was found that UUU coded for the amino acid phenylalanine by creating mRNA entirely of uracil. The mRNA
The Nirenberg and Matthaei experiment was a scientific experiment performed on May 15, 1961, by Marshall W. Nirenberg and his post-doctoral fellow, Heinrich J. Matthaei. The experiment cracked the genetic code by using nucleic acids, tRNA, and amino acids to translate specific polypeptide chain.
In the experiment, they prepared an extract from bacterial cells that could make protein even when no intact living cells were present. Adding an artificial form of RNA, poly-U, to this extract caused it to make a protein composed entirely of the amino acid phenylalanine. This experiment cracked the first codon of the genetic code and showed that RNA controlled the production of specific types of protein.
Nirenberg was awarded the 1968 Nobel Prize in Physiology or Medicine.
Nirenberg later worked with Phillip Leder and performed an experiment to determine the triplet nature of the genetic code and allowed the remaining ambiguous codons in the genetic code to be deciphered.
(UUU..UUU….) added it to a test tube with amino acids, ribosomes, RNA polymerase and other needed materials. It resulted in a protein made of only phenylalanine. Further research determined the rest of the code.

12. Genetic Code
The code has redundancy (GGU, GGC, GGA, and GGG); all code for the amino acid glycine.
Each codon only codes for one amino acid.
The code is a universal code meaning almost all cells use the same code. A eukaryotic gene can be expressed in a prokaryotic cell.
It is called the universal genetic code BUT there are several exceptions where UAA and UAG represent glutamine instead of stop in a species of paramecia. Animal mitochondria use AUA for methionine instead of isoleucine and vertebrate mitochondria use AGA and AGG as stop codons.

13. Specifying or Coding for a Polypeptide
This gene designates that the following peptide chain be made with the amino acids in this particular order.

14. Transcription Overview
Transcription-RNA synthesis from a DNA template
Initiation
Elongation
Termination
RNA processing

15. Initiation
Initiation-There is a region prior to beginning of a gene where the RNA polymerase attaches called the promoter region. The promoter region determines which side of the gene will be transcribed. In a prokaryotic cell, the RNA polymerase attaches directly to the region, but in a eukaryotic cell there are transcription factors (proteins) which help facilitate the attachment of the RNA polymerase. Within the promoter region, there is a sequence of TATA nucleotides, called the TATA box, that helps identify where the RNA polymerase should bind. Once the RNA polymerase attaches, there are even more transcription factors that attach. Now the RNA polymerase unwinds the DNA at the start point of the gene.
In prokaryotes there is only one type of RNA polymerase, but in eukaryotes there are three types of RNA polymerase.

16. Elongation
B. Elongation- RNA polymerase unwinds the DNA and base pairs RNA nucleotides to the DNA gene. RNA is made 5’ → 3’ so the DNA gene is 3’ →5’.
The base pairing for RNA is adenine with uracil and guanine with cytosine. The approximate rate of base paring by RNA polymerase is about 60 RNA nucleotides/minute. The RNA molecule will peel off of the DNA gene and DNA molecule will reform. The average mRNA is 8000 base pairs long. A gene can be simultaneously transcribed by a number of transcription factors. This is important when many copies of the same protein are needed, such as albumin in an egg, or hemoglobin in a red blood cell.
Do the math, if the average protein is 400 amino acids long then the number of nucleotides absolutely necessary to code for an average protein is 1200 nucleotides. However the average mRNA is 8000 base pairs long. There seems to be some extra nucleotides.
Elongation- RNA polymerase unwinds the DNA and base pairs RNA nucleotides to the DNA gene. RNA is made 5′ → 3′ so the DNA gene is 3′ → 5′.

17. Termination 1. Intrinsic termination 2. Extrinsic termination
Animation
RNA synthesis proceeds until the RNA polymerase encounters a sequence that triggers its dissociation. This process is not well understood in eukaryotes. In eukaryotic cells, the RNA polymerase actually passes the termination point before the RNA molecule is released.
C. Termination --there are two different methods for prokaryotic cells
Intrinsic termination -- RNA transcription stops when the newly synthesized RNA molecule forms a G-C-rich hairpin loop followed by a run of U’s. When the hairpin forms, the mechanical stress breaks the weak rU-dA bonds. This pulls the poly-U transcript out of the active site of the RNA polymerase, in effect, terminating transcription.
Extrinsic termination -- a protein factor called rho destabilizes the interaction between the template and the mRNA, thus releasing the newly synthesized mRNA from the elongation complex.

18. RNA Processing Eukaryotic RNA processing 5' cap is added.
At the 3' end adenine nucleotides are added (poly-A-tail).
Introns are removed
D. RNA processing- In eukaryotic cells the RNA is processed.
5' cap with a modified guanine nucleotide is added.
At the 3' end adenine nucleotides are added (poly-A-tail).
These modifications prevent the mRNA from being degraded
Signal the ribosome where to attach.
The poly-A-tail also determines how many times the mRNA can be translated before it is destroyed.
The average immature RNA is 8000 nucleotides long but the mature mRNA is 1200 nucleotides long. There are noncoding regions (introns) that are removed in eukaryotic cells. The remaining regions (exons) are joined together to form the cistron.

19. Removing Introns A spliceosome removes the introns.
Spliceosomes are composed of smaller particles called snRNP (made of proteins and snRNA).
The spliceosome will splice the intron at a specific RNA sequence releasing a "lariat" RNA.

20. RNA Processing
Graphic
Different exons are recombined in different ways for certain mRNAs. This increases the number of different proteins.

21. Exon Shuffling and Different Proteins
Proteins often have a modular architecture consisting of discrete regions called domains
In many cases, different exons code for the different domains in a protein
Exon shuffling may result in the evolution of new proteins.

22. Ready for Translation
This mRNA has been processed and is called mature mRNA. It is ready to go to the cytoplasm for translation.

23. Differences in Protein Synthesis between Prokaryotes and Eukaryotes
The major difference between prokaryotic and eukaryotic protein synthesis is prokaryotes do not have a nucleus so transcription and translation can be simultaneous.
Also, the mRNA is not processed like eukaryotic RNA.
Both types of cells use the same genetic code.
Prokaryotes do not have introns like eukaryotes.
RNA in prokaryotes does not have to be processed like eukaryotes.
Transcription and translation can be simultaneous in prokaryotes.

24. Protein Syntheis: A Review
This shows the result of a base pair insertion or deletion substitution. Results in frame shift mutation. The first one results in missense protein.
The second example results in an immediate stop codon resulting in immediate nonsense.
The last example results in three missing nucleotides which means that the one amino acid is missing and no frameshift has been caused.
Example of a frameshift mutation
Tay-Sachs disease is an autosomal recessive disorder marked by degeneration of both mental and physical capabilities. At its worst, death usually occurs around the age of four, although there are juvenile and late-onset forms of the disease. It is caused by the build-up of a particular lipid in the brain, that is normally broken down by hexosaminidase. Though different mutations in the same gene can lead to the disease, in most cases, a 4 base pair insertion in of the HEXA gene (chromosome 15) renders a premature stop codon, leading to a profound deficiency of one of the subunits of hexosaminidase protein.
It is important to note that not all frameshift and point mutations result in unstable, inactive proteins. Indeed, frameshift mutations can lead to new genes and novel proteins. After all, it is the ability to change DNA that leads to genetic adaptation and, ultimately, evolution of a species. It is important to relate this back to evolution. 24 24

25. Homework
Explaining the role of RNA polymerase and Spliceosomes (snRNPs) in protein synthesis.
Explain the organization of the genetic material in prokaryotes and eukaryotes and contrast the process of Transcription in prokaryotes and eukaryotes