structural and regulatory genes constitute the genotype, that is, the complete genetic informational caparity of an organism. Genes determine all hereditary traits, and they control all the potential activities that can take place within living cells.

Genes are sequences of DNA that have specific

functions.

Bacterial cells usually have a single set of genes and are said to be haploid. In contrast, eukaryotic microorganisms generally have pairs of matching chromosomes and are diploid, having two copies of each gene during at least part of their life cycles. Some eukaryotic microorganisms are haploid during part of their life cycle. When both copies of the gene are identical [aɪ'dentɪk(ə)l , the cell is homozygous. When the cor­responding copies of the gene differ, the cell is heterozygous. For example, eukaryotic microorganisms with homozygous genes for color may appear red or white; those with heterozygous genes for red and

206 CHAPTER 7 MICROBIAL GENETICS: REPLICATION AND EXPRESSION OF GENETIC INFORMATION

One Gene—One Polypeptide

At the turn of the twentieth century, a physician named Archibald Garrod was studying human metabolic dis­orders that seemed to run in families. He hypothesized that the specific units of inheritance must function through the synthesis of specific enzymes. This hypoth­esis received strong support from the studies reported by George Beadle and Edward Tatum in 1941 on the re­lationship between genes—the units of inheritance— and the metabolism of the bread mold Neurospora crassa (see Figure). Prototrophic strains of N. crassa can grow on a minimal medium containing only sucrose, mineral salts, and biotin. From these substances, N. crassa can synthesize all of its other nutritional requirements, in­cluding the amino acids needed for making proteins. This fungus made an excellent choice as the experimen­tal organism to study gene function because much was already known about some of its metabolic pathways. In particular each of the enzymatic steps in the biosyn­thetic pathway for making the amino acid arginine was known.

Beadle and Tatum designed experiments to detect mutations that produced auxotrophs that could not syn­thesize arginine. They then analyzed these mutants to see which metabolic step in the synthesis of arginine was affected. To increase the frequency of mutations, Beadle and Tatum X-rayed Neurospora spores. The prog­

eny were allowed to grow on a complete medium, which contained all necessary metabolites, including arginine. Next, they tested the abilities of the progeny to grow on a minimal medium lacking amino acids to see if mutations had occurred that resulted in the inability to synthesize arginine. An auxotrophic mutant that could not synthesize arginine would grow on the com­plete medium but not on the minimal medium.

Beadle and Tatum hypothesized that if genes (hered­itary units) controlled the production of specific en­zymes, they could detect mutants that could not make different specific enzymes and so could be blocked at different steps in the metabolic pathway for arginine biosynthesis. In fact, Beadle and Tatum identified and isolated many such mutants.

Analysis of cell extracts of mutants revealed a differ­ent defective enzyme in each mutant strain. For each en­zyme in the arginine biosynthetic pathway, Beadle and Tatum were able to isolate a mutant strain with a defec­tive form of the enzyme needed for that step. They thus provided evidence favoring the "one gene, one en­zyme" hypothesis. Other scientists continued to study the relationship between genes and proteins, modifying the original hypothesis slightly, reaching the conclusion that genes code for polypeptides, the chains of amino acids that make up proteins.

white may appear red or white if one gene is prefer­entially expressed (dominant) over the other (reces­sive) or pink if both genes are simultaneously ex­pressed (codominant).

RNA Synthesis

The transfer of information from DNA to protein is accomplished in two stages (FIG. 7-14). The informa­tion in the DNA molecule is initially used to direct the synthesis of ribonucleic acid (RNA) molecules in a process called transcription. Transcription is so named because the information in the DNA is effec­tively copied or transcribed into RNA. In the second stage, called translation, the RNA directs the synthe­sis of proteins. Translation is so named because the order of nucleotides in RNA is translated into the or­der of amino acids of proteins. Transcription and translation occur in tandem in prokaryotic cells.

FIG. 7-14 The information in DNA is transferred to RNA during transcription. The RNA then directs the synthesis of proteins during translation.

The one gene-one hypothesis was experimentally demonstrated by Beadle and Tatum. They studied the fungus Neurospora, which forms spores that have single sets of genes. They were able to collect in­dividual spores and to culture them so that they could observe any changes in the genes of the fungus that altered its nutritional requirements. Mutants, which were unable to carry out complete biosyn­thetic pathways, could grow on complete media but not on minimal media. The minimal media lacked the nutrients required for growth that the mutants could no longer synthesize. Growth on minimal me­dia with specific compounds added, such as vitamins and amino acids, enabled them to determine which compounds the mutant fungi could not synthesize. In this manner they were able to identify the genetic changes that occurred and to associate specific genes with specific metabolic activities.

FIG. 7-15 Ribonucleic acid (RNA) is composed of ribonucleotides that have the sugar ribose, a phosphate group, and one of four nucleic acid bases: uracil, cytosine, adenine, or guanine.

However, in eukaryotic cells, transcription occurs in the nucleus and translation subsequently occurs on the ribosomes in the cytoplasm. Therefore, in eu­karyotic cells, transcription and translation are sepa­rated in time and space.

RNA acts as an informational mediator between the DNA where genetic information is stored and the proteins that functionally express that information.

Ribonucleic Acid (RNA)—Functions and Types

RNA differs from DNA in several respects (FIG. 7-15). The 5-carbon sugar in the RNA nucleotide is ri- bose, which has one more oxygen atom than the de- oxyribose in DNA. One of RNA's nitrogenous bases is uracil instead of thymine. Also, RNA is usually single-stranded.

RNA contains ribose, adenine, uracil, cytosine, and guanine.

Three different kinds of RNA occur in all living cells. They are ribosomal RNA, messenger RNA, and transfer RNA. Each of these RNA molecules has dif­ferent functions and different physical-chemical properties (Table 7-1). Ribosomal RNA (rRNA) is an important structural component of ribosomes, the sites where proteins are synthesized within cells. Messenger RNA (mRNA) carries the information from the DNA molecule to the ribosome. At the ribo­somes, protein synthesis actually occurs when the in­formation encoded in the mRNA molecule is used to specify the sequence of amino acids that comprise the

protein. The mRNA contains the genetic information of DNA in a single-stranded molecule complemen­tary in base sequence to a portion of the base se­quence of DNA. The mRNA sequence directs incor­poration of amino acids into a growing polypeptide in the process called translation, which takes place on the surface of the ribosome. Transfer RNA (tRNA) molecules help align amino acids during protein syn­thesis in the order specified by an mRNA molecule. Transfer RNAs form a bridge between mRNA and an amino acid, thereby transferring the genetic informa­tion carried by the mRNA to the amino acid sequence of the polypeptide. Working together, the three types of molecules convert genetic information from the language ['læŋgwɪʤ] of nucleotides to the language of amino acids, the building blocks of proteins.

Three types of RNA molecules (rRNA, mRNA, and tRNA) are involved in the transfer of information from DNA to proteins.

Transcription

Transcription is the process by which information stored in the DNA molecule is used to code for the synthesis of RNA (FIG. 7-16). During transcription, RNA nucleotides in association with RNA polymerase pair with complementary DNA bases. The base pairs between DNA and RNA are: thymine (DNA) and adenine (RNA), adenine (DNA) and: uracil (RNA), guanine (DNA) and cytosine (RNA), and cytosine (DNA) and guanine (RNA). Once the RNA bases are properly aligned in the order sped-

where transcription begins is called the promoter re­gion. There are different promoters for the initiation of transcription of different genes. The promoter de­termines the site of transcription initiation and which of the two DNA strands is to serve as the sense strand for transcription in that region.

Transcription begins in the promoter region of DNA

where RNA polymerase binds.

The RNA polymerase moves along the DNA tem­plate, synthesizing a new strand of RNA until a spe­cific termination sequence is reached. Then the RNA polymerase falls off the sense strand of the DNA and transcription stops.

Synthesis of mRNA in Prokaryotic and Eukaryotic Cells—Split Genes

There is a fundamental difference in how mRNA is formed in prokaryotic and eukaryotic cells. The sequence of nucleotides in the messenger RNA molecule of prokaryotic cells corresponds exactly with the sequence of nucleotides in the DNA. In con­trast, the RNA molecules of eukaryotic microorgan­isms ai generally extensively modified after tran­scription from the DNA to form mRNA (FIG. 7-18).

Unlike the genes of bacterial cells, the DNA se­quences coding for RNA molecules in eukaryotic cells is not continuous. There are intervening DNA sequences, called introns, between the nucleotide se­quences that actually constitute the gene. The se­quences that constitute the gene are called exons. Hence, the genes of eukaryotic cells are said to be split genes. When DNA is transcribed the RNA ini­tially contains both introns and exons. This precursor of messenger RNA in eukaryotes, known as hnRNA (heterogeneous nuclear RNA), is then subjected to substantial post-transcriptional modification within the nucleus. This post-transcriptional modification removes the introns and forms a messenger RNA that only has exons.

Protein Synthesis—Translation of the Genetic Code

mRNA and the Genetic Code

The information in mRNA specifies the sequence of amino acids in the protein made during translation. The translation of the information in the mRNA mol­ecule, that is, reading mRNA, is a directional process. Messenger RNA is read in a 5'-P —> 3'-OH direction. The polypeptide that is made is synthesized from the amino terminal to the carboxyl terminal end. Here, we see the importance of having a mechanism for recognizing direction in informational macromole­cules. Just as we have the convention for reading the English language from left to right, the correct inter­pretation of the information stored in the mRNA

FIG. 7-18 In eukaryotic cells the primary transcript (hnRNA) is extensively modified within the nucleus to produce mRNA. The conversion of hnRNA to mRNA in­volves the removal of introns. Each mRNA usually encodes only a single gene. In prokaryotic cells the primary tran­script serves as the mRNA. It often encodes several genes.

molecule requires that it be read from the 5'-P —» 3'-OH free end.

The information in mRNA specifies the sequence of amino acids in a protein during translation.

Within mRNA, three sequential nucleotides are used to code for a given amino acid. The genetic code, therefore, is termed a triplet code. Each of the triplet nucleotide sequences is called a codon. Adding or deleting nucleotides causes mutations be­cause the codon changes (FIG. 7-19). The genetic code is said to be degenerate because more than one codon can specify the same amino acid. The mRNA mole­cule is read one codon at a time. In other words, the three nucleotides that specify a single amino acid are read together. There are no spaces between the codons that are read. Therefore establishing a read­ing frame is critical for extracting the proper infor­mation. Adding or deleting a single base pair in the DNA changes the reading frame of the transcribed messenger RNA. Such frame-shift mutations can re­sult in the misreading of large numbers of codons (FIG. 7-20), thus producing proteins that are inactive because they have the wrong amino acid sequence.

Three nucleotides constitute a codon that specifies an amino acid.

FIG. 7-19 Deletion mutations occur when one or more nucleotides are omitted during DNA replication. Addition mutations occur when one or more nucleotides are added dur­ing DNA replication.

FIG. 7-20 A frame shift mutation results from nucleotide deletions or additions.

Each codon consists of three nucleotides. There are (our different nucleotides, making possible 64 codons. The four different nucleotides in different three-base combinations lead to 4 X 4 X 4 = 64 pos­sibilities (Table 7-2, p. 212). The genetic language, which is almost universal, can therefore be said to have four letters in the alphabet and 64 words in the dictionary, each word containing three letters.

Proteins in biological systems normally contain only 20 L-amino acids. Thus there are many more codons than are strictly needed for the translation of genetic information into functional proteins. More than one codon can code for the insertion of the same amino acid into the polypeptide chain. Consequently, a silent mutation can occur in which there is an alter­ation in the nucleotide sequence without a change in the amino acid sequence. In such cases, changes in genotype would not be reflected in changes in phe­notype.

Additionally, there are three codons that do not I code for any amino acid. These codons have been re­ferred to as nonsense codons. Actually, nonsense codons serve a very important function, acting like the period at the end of a sentence and signalling ter­mination of synthesis of a polypeptide chain.

More than one codon can code for the same amino acid. The genetic code is degenerate.

tRNA and Polypeptide Formation

Translation of the genetic code into protein molecules occurs at the ribosomes. Ribosomes provide the spa­tial framework and structural support for aligning the translational process of protein synthesis. Distor­tion of the proper configuration of the ribosome can prevent proper information exchange and expression of the genetic information. This forms the basis for the action of many antibiotics, such as erythromycin.

Transfer RNA (tRNA) attaches to amino acids and brings them to the ribosomes. There is a specific tRNA for each amino acid. In addition to bringing the amino acids to the ribosomes, the tRNA also properly aligns them during translation (FIG. 7-21). Each tRNA molecule contains a specific anticodon, a three-base nucleotide sequence that is complemen­tary to the three-base nucleotide sequence of the codon. The pairing of the codons of the mRNA mol­ecules with the anticodons of the tRNA molecules determines the order of amino acid sequence in the polypeptide chain. The third base of the anticodon does not always properly recognize the third base of