PROTEIN SYNTHESIS Table of Contents One-gene-one-protein | The structure of hemoglobin | Viruses contain DNA RNA links the information

in DNA to the sequence of amino acids in protein Transcription: making an RNA copy of a DNA sequence | The Genetic Code Protein Synthesis | Mutations redefined | Links One-gene-one-protein | Back to Top During the 1930s, despite great advances, geneticists had several frustrating questions yet to answer: What exactly are genes? How do they work? What produces the unique phenotype associated with a specific allele? Answers from physics, chemistry, and the study of infectious disease gave rise to the field of molecular biology. Biochemical reactions are controlled by enzymes, and often are organized into chains of reactions known as metabolic pathways. Loss of activity in a single enzyme can inactivate an entire pathway. Archibald Garrod, in 1902, first proposed the relationship through his study of alkaptonuria and its association with large quantities "alkapton". He reasoned unaffected individuals metabolized "alkapton" (now called homogentistic acid) to other products so it would not buildup in the urine. Garrod suspected a blockage of the pathway to break this chemical down, and proposed that condition as "an inborn error of metabolism". He also discovered alkaptonuria was inherited as a recessive Mendelian trait. George Beadle and Edward Tatum during the late 1930s and early 1940s established the connection Garrod suspected between genes and metabolism. They used X rays to cause mutations in strains of the mold Neurospora. These mutations affected a single genes and single enzymes in specific metabolic pathways. Beadle and Tatum proposed the "one gene one enzyme hypothesis" for which they won the Nobel Prize in 1958.

Since the chemical reactions occurring in the body are mediated by enzymes, and since enzymes are proteins and thus heritable traits, there must be a relationship between the gene and proteins. George Beadle, during the 1940s, proposed that mutant eye colors in Drosophila was caused by a change in one protein in a biosynthetic pathway. In 1941 Beadle and coworker Edward L. Tatum decided to examine step by step the chemical reactions in a pathway. They used Neurospora crassa as an experimental organism. It had a short life-cycle and was easily grown. Since it is haploid for much of its life cycle, mutations would be immediately expressed. The meiotic products could be easily inspected. Chromosome mapping studies on the organism facilitated their work. Neurospora can be grown on a minimal medium, and it's nutrition could be studied by its ability to metabolize sugars and other chemicals the scientist could add or delete from the mixture of the medium. It was able to synthesize all of the amino acids and other chemicals needed for it to grow, thus mutants in synthetic pathways would easily show up. X-rays induced mutations in Neurospora, and the mutated spores were placed on growth media enriched with all essential amino acids. Crossing the mutated fungi with non-mutated forms produced spores which were then grown on media supplying only one of the 20 essential amino acids. If a spore lacked the ability to synthesize a particular amino acid, such as Pro (proline), it would only grow if the Proline was in the growth medium. Biosynthesis of amino acids (the building blocks of proteins) is a complex process with many chemical reactions mediated by enzymes, which if mutated would shut down the pathway, resulting in no-growth. Beadle and Tatum proposed the "one gene one enzyme" theory. One gene codes for the production of one protein. "One gene one enzyme" has since been modified to "one gene one polypeptide" since many proteins (such as hemoglobin) are made of more than one polypeptide.

The Beadle and Tatum experiment that suggested the one gene one enzyme hypothesis. Images from Purves et al., Life: The Science of Biology, 4th Edition, by Sinauer Associates (www.sinauer.com) and WH Freeman (www.whfreeman.com), used with permission. The Structure of Hemoglobin | Back to Top Linus Pauling used electrophoresis to separate hemoglobin molecules. Sickle-cell anemia (h) is a recessive allele in which a defective hemoglobin is made, ultimately causing pain and death to those individuals homozygous recessive for the trait. Pauling reasoned that if Beadle and Tatum were correct, there should be a slight (but detectable) difference between the structure of a normal (HH) and sickle cell (hh) hemoglobin due to genetic differences. Heterozygotes (Hh, also sampled by Pauling) make both normal and "sickle cell" hemoglobins. Later, Vernon Ingram discovered that the normal and sickle-cell hemoglobins differ by only 1 (out of a total of 300) amino acids. Viruses Contain DNA | Back to Top The coats of viruses act as antigens, initiating an antigen-specific antibody response. Remember that vaccines work by either prompting the immune system to make antibodies or by supplying antibodies. If a virus (or anything else for that matter) mutates its antigens, the immune system is forever playing catch-up.

RNA Links the Information in DNA to the Sequence of Amino Acids in Protein | Back to Top Ribonucleic acid (RNA) was discovered after DNA. DNA, with exceptions in chloroplasts and mitochondria, is restricted to the nucleus (in eukaryotes, the nucleoid region in prokaryotes). RNA occurs in the nucleus as well as in the cytoplasm (also remember that it occurs as part of the ribosomes that line the rough endoplasmic reticulum). Scientists for some time had suspected such a link between DNA and proteins. Cells of developing embryos contain high levels of RNA. Rapidly growing E. coli has half its mass as ribosomes. Ribosomes are 2/3 RNA (a type of RNA known as ribosomal RNA or rRNA) and 1/3 protein. RNA is synthesized from viral DNA in an infected cell before protein synthesis begins. Some viruses, for example Tobacco Mosaic Virus (TMV) have RNA in place of DNA. If RNA extracted from a virus was injected into a host cell the cell began to make new viruses. Clearly RNA was involved in protein synthesis. Crick's central dogma. Information flow (with the exception of reverse transcription) is from DNA to RNA via the process of transcription, and thence to protein via translation. Transcription is the making of an RNA molecule off a DNA template. Translation is the construction of an amino acid sequence (polypeptide) from an RNA molecule. Although originally called dogma, this idea has been tested repeatedly with almost no exceptions to the rule being found (save retroviruses).

The central dogma. Image from Purves et al., Life: The Science of Biology, 4th Edition, by Sinauer Associates (www.sinauer.com) and WH Freeman (www.whfreeman.com), used with permission. The blue-background graphics throughout this chapter are from the University of Illinois' DNA and Protein Synthesis site.

Messenger RNA (mRNA) is the blueprint for construction of a protein. Ribosomal RNA (rRNA) is the construction site where the protein is made. Transfer RNA (tRNA) is the truck delivering the proper amino acid to the site at the right time.

RNA has ribose sugar instead of deoxyribose sugar. The base uracil (U) replaces thymine (T) in RNA. Most RNA is single stranded, although tRNA will form a "cloverleaf" structure due tocomplementary base pairing.

Transcription: making an RNA copy of a DNA sequence | Back to Top RNA polymerase opens the part of the DNA to be transcribed. Only one strand of DNA (the template strand) is transcribed. RNA nucleotides are available in the region of the chromatin (this process only occurs during Interphase) and are linked together similar to the DNA process.

The Genetic Code: Translation of RNA code into protein | Back to Top The code consists of at least three bases, according to astronomer George Gamow. To code for the 20 essential amino acids a genetic code must consist of at least a 3-base set (triplet) of the 4 bases. If one considers the possibilities of arranging four things 3 at a time (4X4X4), we get 64 possible code words, or codons (a 3-base sequence on the mRNA that codes for either a specific amino acid or a control word). The genetic code was broken by Marshall Nirenberg and Heinrich Matthaei, a decade after Watson and Crick's work. Nirenberg discovered that RNA, regardless of its source organism, could initiate protein synthesis when combined with contents of broken E. coli cells. By adding poly-U to each of 20 test-tubes (each tube having a different "tagged" amino acid) Nirenberg and Matthaei were able to determine that the codon UUU (the only one in poly-U) coded for the amino acid phenylalanine.

Steps in breaking the genetic code: the deciphering of a poly-U mRNA. Image from Purves et al., Life: The Science of Biology, 4th Edition, by Sinauer Associates (www.sinauer.com) and WH Freeman (www.whfreeman.com), used with permission. Likewise, an artificial mRNA consisting of alternating A and C bases would code for alternating amino acids histidine and threonine. Gradually, a complete listing of the genetic code codons was developed.

Deciphering the code: poly CA. Image from Purves et al., Life: The Science of Biology, 4th Edition, by Sinauer Associates (www.sinauer.com) and WH Freeman (www.whfreeman.com), used with permission. The genetic code consists of 61 amino-acid coding codons and three termination codons, which stop the process of translation. The genetic code is thus redundant (degenerate in the sense of having multiple states amounting to the same thing), with, for example, glycine coded for by GGU, GGC, GGA, and GGG codons. If a codon is mutated, say from GGU to CGU, is the same amino acid specified?

The genetic code. Image from Purves et al., Life: The Science of Biology, 4th Edition, by Sinauer Associates (www.sinauer.com) and WH Freeman (www.whfreeman.com), used with permission. Protein Synthesis | Back to Top Prokaryotic gene regulation differs from eukaryotic regulation, but since prokaryotes are much easier to work with, we focus on prokaryotes at this point. Promoters are sequences of DNA that are the start signals for the transcription of mRNA. Terminators are the stop signals. mRNA molecules are long (500- 10,000 nucleotides). Ribosomes are the organelle (in all cells) where proteins are synthesized. They consist of two-thirds rRNA and one-third protein. Ribosomes consist of a small (in E. coli ,

30S) and larger (50S) subunits. The length of rRNA differs in each. The 30S unit has 16S rRNA and 21 different proteins. The 50S subunit consists of 5S and 23S rRNA and 34 different proteins. The smaller subunit has a binding site for the mRNA. The larger subunit has two binding sites for tRNA.

Subunits of a ribosome. Image from Purves et al., Life: The Science of Biology, 4th Edition, by Sinauer Associates (www.sinauer.com) and WH Freeman (www.whfreeman.com), used with permission. Transfer RNA (tRNA) is basically cloverleaf-shaped. tRNA carries the proper amino acid to the ribosome when the codons call for them. At the top of the large loop are three bases, theanticodon, which is the complement of the codon. There are 61 different tRNAs, each having a different binding site for the amino acid and a different anticodon. For the codon UUU, the complementary anticodon is AAA. Amino acid linkage to the proper tRNA is controlled by the aminoacyl-tRNA synthetases. Energy for binding the amino acid to tRNA comes from ATP conversion to adenosine monophosphate (AMP).

Two models of tRNA. Image from Purves et al., Life: The Science of Biology, 4th Edition, by Sinauer Associates (www.sinauer.com) and WH Freeman (www.whfreeman.com), used with permission. Translation is the process of converting the mRNA codon sequences into an amino acid sequence. The initiator codon (AUG) codes for the amino acid Nformylmethionine (f-Met). No transcription occurs without the AUG codon. f-Met is always the first amino acid in a polypeptide chain, although frequently it is removed after translation. The intitator tRNA/mRNA/small ribosomal unit is called the initiation complex. The larger subunit attaches to the initiation complex. After the initiation phase the message gets longer during the elongation phase.

Translation. Image from Purves et al., Life: The Science of Biology, 4th Edition, by Sinauer Associates (www.sinauer.com) and WH Freeman (www.whfreeman.com), used with permission. New tRNAs bring their amino acids to the open binding site on the ribosome/mRNA complex, forming a peptide bond between the amino acids. The complex then shifts along the mRNA to the next triplet, opening the A site. The new tRNA enters at the A site. When the codon in the A site is a termination codon, a releasing factor binds to the site, stopping translation and releasing the ribosomal complex and mRNA.

Termination. Image from Purves et al., Life: The Science of Biology, 4th Edition, by Sinauer Associates (www.sinauer.com) and WH Freeman (www.whfreeman.com), used with permission. Often many ribosomes will read the same message, a structure known as a polysome forms. In this way a cell may rapidly make many proteins.

Many ribosomes translating the same message, a polysome. Image from Purves et al., Life: The Science of Biology, 4th Edition, by Sinauer Associates (www.sinauer.com) and WH Freeman (www.whfreeman.com), used with permission. The illustration below is from Genentech's Access Excellence site, which may be reeached by clicking here. The drawing is available at http://www.gene.com/ae/AB/GG/protein_synthesis.html

Mutations Redefined | Back to Top We earlier defined mutations as any change in the DNA. We now can refine that definition: a mutation is a change in the DNA base sequence that results in a change of amino acid(s) in the polypeptide coded for by that gene. Alleles are alternate sequences of DNA bases (genes), and thus at the molecular level the products of alleles differ (often by only a single amino acid, which can have a ripple effect on an organism by changing ). Addition, deletion, or addition of nucleotides can alter the

polypeptide. Point mutations are the result of the substitution of a single base. Frameshift mutations occur when the reading frame of the gene is shifted by addition or deletion of one or more bases. With the exception of mitochondria, all organisms use the same genetic code. Powerful evidence for the common ancestry of all living things. Links | Back to Top

The Genetic Code

The genetic code consists of 64 triplets of nucleotides. These triplets are called codons.With three exceptions, each codon encodes for one of the 20 amino acids used in the synthesis of proteins. That produces some redundancy in the code: most of the amino acids being encoded by more than one codon. One codon, AUG serves two related functions:

Index to this page

The RNA Codons The DNA Codons Codon Bias Exceptions to the Code

it signals the start of translation it codes for the incorporation of the amino acid methionine (Met) into the growing polypeptide chain

The genetic code can be expressed as either RNA codons or DNA codons. RNA codons occur in messenger RNA (mRNA) and are the codons that are actually "read" during the synthesis of polypeptides (the process calledtranslation). But each mRNA molecule acquires its sequence of nucleotides by transcription from the corresponding gene. Because DNA sequencing has become so rapid and because most genes are now being discovered at the level of DNA before they are discovered as mRNA or as a protein product, it is extremely useful to have a table of codons expressed as DNA. So here are both. Note that for each table, the left-hand column gives the first nucleotide of the codon, the 4 middle columns give the second nucleotide, and the last column gives the third nucleotide.
The RNA Codons
Second nucleotide

U UUU Phenylalanine (Phe) UUC Phe U UUA Leucine (Leu) UUG Leu CUU Leucine (Leu) CUC Leu C CUA Leu CUG Leu AUU Isoleucine (Ile) AUC Ile A AUA Ile AUG Methionine (Met) or START GUU Valine Val GUC (Val) G GUA Val GUG Val GCA Ala GCG Ala CCA Pro CCG Pro UCA Ser UCG Ser

C UCU Serine (Ser) UCC Ser

A UAU Tyrosine (Tyr) UAC Tyr UAA STOP UAG STOP CAU Histidine (His) CAC His CAA Glutamine (Gln) CAG Gln

G UGU Cysteine (Cys) UGC Cys UGA STOP U C A

UGG Tryptophan (Trp) G CGU Arginine (Arg) CGC Arg CGA Arg CGG Arg U C A G U C A G

CCU Proline (Pro) CCC Pro

ACU Threonine (Thr) AAU Asparagine (Asn) AGU Serine (Ser) ACC Thr ACA Thr ACG Thr AAC Asn AAA Lysine (Lys) AAG Lys GAU Aspartic acid (Asp) GAC Asp GAA Glutamic acid (Glu) GAG Glu AGC Ser AGA Arginine (Arg) AGG Arg

GCU Alanine (Ala) GCC Ala

GGU Glycine (Gly) GGC Gly GGA Gly GGG Gly

U C A G

The DNA Codons

These are the codons as they are read on the sense (5' to 3') strand of DNA. Except that the nucleotide thymidine (T) is found in place of uridine (U), they read the same as

RNA codons. However, mRNA is actually synthesized using the antisense strand of DNA (3' to 5') as the template. [Discussion] This table could well be called the Rosetta Stone of life. The Genetic Code (DNA)
TTT Phe TTC Phe TTA Leu TTG Leu CTT Leu CTC Leu CTA Leu CTG Leu ATT Ile ATC Ile ATA Ile TCT Ser TAT Tyr TCC Ser TAC Tyr TGT Cys TGC Cys

TCA Ser TAA STOP TGA STOP TCG Ser TAG STOP TGG Trp CCT Pro CAT His CCC Pro CAC His CCA Pro CAA Gln CCG Pro CAG Gln ACT Thr AAT Asn ACC Thr AAC Asn ACA Thr AAA Lys CGT Arg CGC Arg CGA Arg CGG Arg AGT Ser AGC Ser AGA Arg AGG Arg GGT Gly GGC Gly GGA Gly GGG Gly

ATG Met* ACG Thr AAG Lys GTT Val GTC Val GTA Val GTG Val GCT Ala GAT Asp GCC Ala GAC Asp GCA Ala GAA Glu GCG Ala GAG Glu

*When within gene; at beginning of gene, ATG signals start of translation.

Codon Bias

All but two of the amino acids (Met and Trp) can be encoded by from 2 to 6 different codons. However, the genome of most organisms reveals that certain codons are preferred over others. In humans, for example, alanine is encoded by GCC four times as often as by GCG. This probably reflects a greater translation efficiency by the translation apparatus (e.g., ribosomes) for certain codons over their synonyms. [More]
Exceptions to the Code The genetic code is almost universal. The same codons are assigned to the same amino acids and to the same START and STOP signals in the vast majority of genes in animals, plants, and microorganisms. However, some exceptions have been found. Most of these involve assigning one or two of the three STOP codons to an amino acid instead.

Mitochondrial genes When mitochondrial mRNA from animals or microorganisms (but not from plants) is placed in a test tube with the cytosolic protein-synthesizing machinery (amino acids, enzymes, tRNAs, ribosomes) it fails to be translated into a protein. The reason: these mitochondria use UGA to encode tryptophan (Trp) rather than as a chain terminator. When translated by cytosolic machinery, synthesis stops where Trp should have been inserted. In addition, most

animal mitochondria use AUA for methionine not isoleucine and all vertebrate mitochondria use AGA and AGG as chain terminators. Yeast mitochondria assign all codons beginning with CU to threonine instead of leucine (which is still encoded by UUA and UUG as it is in cytosolic mRNA).

Plant mitochondria use the universal code, and this has permitted angiosperms to transfer mitochondrial genes to their nucleus with great ease.
Link to discussion of mitochondrial genes.

Nuclear genes

Violations of the universal code are far rarer for nuclear genes. A few unicellular eukaryotes have been found that use one or two (of their three) STOP codons for amino acids instead. Nonstandard Amino Acids The vast majority of proteins are assembled from the 20 amino acids listed above even though some of these may be chemically altered, e.g. by phosphorylation, at a later time. However, two cases have been found where an amino acid that is not one of the standard 20 is inserted by a tRNA into the growing polypeptide.

selenocysteine. This amino acid is encoded by UGA. UGA is still used as a chain terminator, but the translation machinery is able to discriminate when a UGA codon should be used for selenocysteine rather than STOP. This codon usage has been found in certain Archaea, eubacteria, and animals (humans synthesize 25 different proteins containing selenium). pyrrolysine. In several species of Archaea and bacteria, this amino acid is encoded by UAG. How the translation machinery knows when it encounters UAG whether to insert a tRNA with pyrrolysine or to stop translation is not yet known.

Prokaryotes and the Operon Model Prokaryotes are sensitive to their environment, and their genetic activity is controlled by specific proteins that interact directly with their DNA to quickly adjust to environmental changes. Genetic expression is the process where genotypes coded in the genes are exhibited by the phenotypes of the individuals. The DNA is copied by the RNA and then synthesized into protein. The process of transcription, which is the synthesis of RNA from a DNA template, is where the regulation of the gene expression is most likely to occur. The default setting for prokaryotes appears to allow for the continual synthesis of protein to occur, whereas in eukaryotes the system is normally off until activated. An operon is a self-regulating series of genes that work in concert. An operon includes a special segment of genes that are regulators of the protein synthesis, but do not code for protein, called the promoter and operator. These segments overlap, and their interaction determines whether the process will start and when it will stop. RNA polymerase must create RNA by moving along the chromosome and reading the genes in the process of transcription. RNA polymerase first attaches to the promoter segment, which signals the beginning of a particular DNA sequence. If not blocked, it passes over the operator and reaches the protein-producing genes where it creates the mRNA that instructs the ribosomes to create the desired protein. This process continues until

the system is blocked by repressor proteins. Repressors bind with the operator and prevent RNA polymerase from proceeding to create mRNA by prohibiting access to the remainder of the proteinproducing genes. As long as the repressor is binding with the operator, no proteins are made. However, when an inducer is present, it binds with the repressor, causing the repressor to change shape and release from the operator. When this happens, the RNA polymerase can proceed with transcription, and protein synthesis begins and continues until another repressor binds with the operator. Refer to the illustration Transcription regulation.

Transcription regulation.

The lac operon model is probably the most studied and well known. In bacteria, such as E. coli, three genes are part of an operon that code for three separate enzymes needed for the breakdown of lactose, a simple sugar. A regulatory gene, located before the operon, continually makes repressor proteins that bind with the operator and prohibit the function of RNA polymerase. The system therefore remains off until a flood of lactose molecules binds with all available repressors and prevents their attachment to the operator. When the operator is free, the production of the enzyme to break down lactose continues until enough of the lactose molecules are broken down to then release repressors to recombine with the operator to stop production of the enzymes. Two additional types of operons exist that operate in the same way except for the function of the operator. The trp operon differs because the repressor is active only when bonded to a specific molecule. For the remainder of the time, it remains unbonded and inactive in the absence of that molecule. Finally, in a positive twist, activators are used by a third type of operon to bond directly with the DNA, which allows the RNA polymerase to work more efficiently. Absent the activators, RNA polymerase proceeds at a slow rate.

Read more: Inheritance: Regulation of Gene Expression in Prokaryotes and Eukaryotes | Infoplease.com http://www.infoplease.com/cig/biology/regulation-gene-expression-prokaryoteseukaryotes.html#ixzz2jZ1b9cuX