Biology 2

CHAPTER 16 Notes

FROM GENE TO PROTEIN

Inherited instructions in DNA direct protein synthesis. Thus, proteins are the links between genotype and phenotype, since proteins are directly involved in the expression of specific phenotypic traits.

I. The study of metabolic defects provided evidence that genes specify proteins:

Science as a process

Archibald Garrod was the first to propose the relationship between genes and proteins (1909).

· He suggested that genes dictate phenotypes through enzymes that catalyze reactions.

· As a physician, Garrod was familiar with inherited diseases, which he called “inborn errors in metabolism.” He hypothesized that such diseases reflect the patient’s inability to make particular enzymes.

· One example he studied was alkaptonuria, which causes the afflicted person’s urine to turn black.

· People with alkaptonuria accumulate alkapton in their urine, causing it to darken on contact with air.

· Garrod reasoned that alkaptonurics, unlike normal individuals, lack the enzyme that breaks down alkapton.

A. How Genes Control Metabolism

Garrod’s hypothesis was confirmed several decades later by research, which determined that specific genes direct production of specific enzymes.

· Biochemists found that cells synthesize and degrade organic compounds visa metabolic pathways, with each sequential step catalyzed by a specific enzyme.

· Geneticists George Beadle and Boris Ephrussi (1930’sZ_ studied eye color in Drosophila. They speculated that mutations affecting eye color block pigment synthesis by preventing enzyme production at certain steps in the pigment synthesis pathway.

George Beadle and Edward Tatum were later able to demonstrate the relationship between genes and enzymes by studying mutants of a bread mold, Neurospora crassa.

· Wild-type Neurospora in laboratory colonies can survive on minimal medium. All other molecules needed by the mold are produced by its own metabolic pathways from this minimal nutrient source.

· Beadle and Tatum searched for mutants or auxotrophs that could not survive on minimal medium because they lacked the ability to synthesize essential molecules.

· Mutants were identified by transferring fragments of growing fungi (in complete medium) to vials containing minimal medium. Fragments that didn’t grow were identified as auxotrophic mutants.

Auxotroph = (Auxo = to augment; troph = nourishment) Nutritional mutants that can only be grown on minimal medium augmented with nutrients not required by the wild type.

Minimal medium = Support medium that is mixed only with molecules required for the growth of wild-type organisms.

· Minimal medium for Neurospora contains inorganic salts, sucrose and the vitamin, biotin.

· Nutritional mutants cannot survive only on minimal medium.

Complete growth medium = Minimal medium supplemented with all 20 amino acids and some other nutrients.

· Nutritional mutants can grow on complete growth medium, since all essential nutrients are provided.

Beadle and Tatum then identified specific metabolic defects (from mutations) by transferring fragments of auxotrophic mutants growing on complete growth medium to vials containing minimal medium each supplemented with only one additional nutrient.

· Vials where growth occurred indicated the metabolic defect, since the single supplement provided the necessary component.

· For example, if a mutant grew on minimal medium supplemented with only arginine, it could be concluded that the mutant was defective in the arginine synthesis pathway.

Experiment:

Beadle and Tatum experimented further to more specifically describe the defect in the multistep pathway that synthesizes the amino acid arginine.

· Arginine synthesis requires three steps each catalyzed by a specific enzyme:

Gene A Gene B Gene C

Enzyme A Enzyme B Enzyme C

Precursor Ornithine Citrulline Arginine

· They distinguished between three classes of arginine auxotrophs by adding either arginine, citrulline, or ornithine to the medium and seeing if growth occurred.

Results:

Some mutants required arginine, some either arginie or citrulline, and others could grow when any of the three were added.

	Mimial Medium (MM)	MM plus Ornithine	MM plus Citrulline	MM plus Arginine
Wild Type Class I Mutants Class II Mutants Class III Mutants	+ - - -	+ + - -	+ + + -	+ + + +

+ = growth, - = no growth

Conclusions:

Beadle and Tatum deduced form their data that the three classes of mutants eaach lacked a different enzyme and were thus blocked at different steps in the arginine synthesis pathway.

· Class I mutants lacked enzyme A; Class II mutants lacked enzyme B; and Class III mutants lacked enzyme C.

· Assuming that each mutant was defective in a single gene, they formulated the one gene-one enzyme hypothesis, which stated that the function of a gene is to dictate the production of a specific enzyme

B. One Gene-One Polypeptide

Beadle and Tatum’s one gene-one enzyme hypothesis has been slightly modified:

· While most enzymes are proteins, many proteins are not enzymes. Proteins that are not enzymes are still, nevertheless, gene products.

· Also, many proteins are comprised of two or more polypeptide chains, each chain specified by a different gene (e.g. globulin chains of hemoglobin).

As a result of this new imformation, Beadle and Tatum’s hypothesis has been restated as one gene-one polypeptide.

II. Transcription and translation are the two main steps from gene to protein: an overview

Ribonucleic acid (RNA) links DNA’s genetic instructions for making proteins to the process of protein synthesis. It copies or transcribes the message from DNA and then translates that message into a protein.

1 RNA, like DNA, is a nucleic acid or polymer of nucleotides.

2 RNA structure differs from DNA in the following ways:

o The five-carbon sugar in RNA nucleotides is ribose rather than deoxyribose.

o The nitrogenous base uracil is found in place of thymine.

o Single strand- may leave nucleus.

The linear sequence of nucleotides in DNA ultimately determines the linear sequence of amino acids in a protein.

1 Nucleic acids are made of four types of nucleotides which differ in their nitrogenous bases. Hundreds or thousands of nucleotides long, each gene has a specific linear sequence of the four possible bases.

2 Proteisn are made of twenty types of amino acids linked in a particular linear sequences (the protein’s primary structure).

3 Information flows from gene to protein through two major processes, transcription and translation.

Transcription = the synthesis of RNA using DNA as a template.

1 A gene’s unique nucleotide sequence is transcribed from DNA to a complementary nucleotide sequence in messenger RNA (mRNA).

2 The resulting mRNA carries this transcript of protein-building instructions to the cell’s protein-synthesizing machinery.

Translation = Synthesis of a polypeptide, which occurs under the direction of messenger RNA (mRNA).

1 During this process, the linear sequence of bases in mRNA is translated into the linear sequence of amino acids in a polypeptide.

2 Translation occurs on ribosomes, complex particles composed of ribosomal RNA (rRNA) and protein that facilitate the orderly linking of amino acids into polypeptide chains.

Prokaryotes and eukaryotes differ in how protein synthesis is organized within their cells.

1 Prokaryotes lack nuclei, so DNA is not segregated from ribosomes or the protein synthesizing machinery. Thus, transcription and translation occur in rapid succession.

2 Eukaryotes have nuclear envelopes that segregate transcription in the nucleus from translation in the cytoplasm; mRNA, the intermediary, is modified before it moves from the nucleus to the cytoplasm; mRNA, the intermediary, is modified before it moves from the nucleus to the cytoplasm where translation occurs. This RNA processing occurs only in eukaryotes.

III. In the genetic code, a particular triplet of nucleotides specifies a certain amino acid: a closer look

There is not a one-to-one correspondence between the nitrogenous bases and the amino acids they specify, since there are only 4 nucleotides and 20 amino acids.

1 A two-to-one correspondance of bases to amnino acids would only specify 16 (4² ) of the 20 amino acids.

2 A three-to-one correspondance of bases to amino acids would specify 64 (4³ ) amino acids.

Researchers have verified that the flow of information from a gene to a protein is based on a triplet code.

· Triplets of nucleotides are the smallest units of uniform length to allow translation into all amino acids with plenty to spare.

· These three-nucleotide "words" are claled codons

Codon = A three-nucleotide seqence in mRNA thatspecifies which amino acid wil be addedt o a growing polypeptide or that signals termination; the basic unit of the gentic code.

Genes are not directly translated ito amino acids but are first trnascrived as codons into mRNA.

· For each gene, only one of the two DNA strans (the template strand) is transcribed.

· The complementary nontemplate strand is the parental strand for making a new template when DNA replicates.

· The same DNA strand can be the template strand for some genes and the nontemplate strand for others.

An mRNA is complementary to the DNA template from which it is transcribed.

· For exmpale, if the triplet nucleotide sequence on teh termplaate DNA strand is CC; GGC, the codon for glycine, will be the complementary mRNA transcript.

· recall that according to the base-pairing rules, uracil (U) in RNa is used in placve of thymine (T); uracil thus base pairs with adenine (A).

During translation, the linar sequence of codons along mRNA is translated into the linear sequence of amino acids in a polypeptide.

· Each mRNA codon specifies whcih one of 20 amino acids willbe incorporated into the corresponding position in a polypeptide.

· Because codons are base triplets, the number of nuceotides making up a genetic mesafe is three times the number of amino acids making up the polypeptide [roduct.

A, Cracking the Genetic Code

The fist codon was deciphered in 1961 by Marshall Nierenberg of the National Is\nstitutes of Health.

· He synthesized an mRNA by linking only uracil-bearign RNa nucleotides, resluting in UU codons.

· Niemberg added this "poly U" to a test-tibe mixture contatining the components necesary for protein synthesis. The artificial mRNA )poly U) was translated into a polypepetide containing a string of only one amino acid, phenylalanine.

· Nirenberg concle\uded that the mRNA codon UUU s[ecifies the amino acd phenylalaline.

· These same techniques were used to determine amiono acids specified by the codons AAA, GGG< and CCC.

More elaborate tenchniques allowed uinvestitgators to determine all 64 codons by the mide 1960's

· 61 of the 64 triplets code for amino acids

· The triplet AUG has a dual function - it is the start signal for translationa nd codes for methionine.

· THree codosn s do not code for amino acids, but signal termination (UAA, UAG and UGA).

There is redundancy in the gentic code, but not ambiguity.

· Redunadncy exists since two or more codons differing only in their thirs base can code for the same amino acid (UUU and UUC both code for phenylalanine(.

· Ambiguity is absent, since codons code for only one amin acid.

The corrct ordering and grouping of nucleotides is importatnt in the molecular language of cells. This ordering is called the reading frame.

Reading frame = The correct grouping of adjacent nucleotide truplets into codons that are in the correct sequence on mRNA/

· FOr example, teh sequence of amino acids -Trp=Phe-Gly-Arg-Phe - can be assembled in the correct sequence and groups.

· The ell reads the message int he correct frame as a series of nooverlapping three letter words: UGG-UUU-GGC_CGU-UUU.

B. The evolutionary Significance of a Common Gentic Language

The genetic code is shared nearly universally among living organisms.

· For exmaple, the RNA codon CCG is translated into proline in all organisms whose genetic codes have neem examined.

· The technology exists to tranfere genes from one species to antoher. For example, the human gene for unsulin can be inserted into be\acteria where it is succesffully expressed.

There are some exeptions to this university:

· Several ciliates (e.g. Paramecium and Tertrahymena) depart from standard code; codons UAA an UaG are not stop signals, bucode for glutamine.

· Mitochondria and chloroplasts have their own DNA that codes for some proteins.

· MItoochondrial genetic codes cary even among organisms;l for example, CUA codes for threonine in yeast mitochndria and leucine in mammalian mitochondria.

The fact that the genetic code is sahred nearly universally by all organisms indicates that this coe was established very early in life's history.

IV. Transcription is the DNA-directed synthesis of RNA; a closer look

Transcription of messenger RNA (mRNA) form template DNA is catylsed by RNA polymerases, which:

· Seperate the two DNA strands and link RNA nucleotides as they base-pari alond the DNA tempalte

· Add nucleotids only to the 3" end, thus. mRNA molecules frow in the 5" to 3" direciton.

These are seceral tupes of RNA polymerase.

· Prolaryotes have only one type of RNA polymeraase that syntheizes all types of RNA - mRNA, rRNA, and tRNA.

· Eukaryotes havge three RNA polymease that transribe genes. RNA polymerase II is the polymerase that catalyzes mRNA synthesis, it trnascribed genes that will be translated into proteins.

Speicific DNA nucleotide sequences mark where transcripto of a gene begins (initiation) and ends (termination) Initiationa dn termination sequences plus the nucleotides in betweenare celled a transaction unit.

Transription unit _ Nucleotide sequece on teh template strand of DNA that is transcribed into a single RNA molecule bny RNa polymease; it includes the intitation and termination seqhences, as well as the nucleotides in between.

· In eukaryotes, a transcritption unit can contatn several genes, so the resulting mRNA may code for differnt, but functionally related, proteins.

Transcription occurs in three key steps: a) polymerase binding and inititation; b) elongation; and c) termination.

A. RNA Poleymerase Binding and Initiation of Transcription

Rna polymerase bind to DNA at regins called promoters.

Promoter - Region of DNA that inclusdes te site where RNA polymerase binds and where transcription begins (initiation site)/ In eukaryotes, the promoter is about 100 nucleotides long and consists of;

a. The intitiation site, where transcription begins.

b. A few nucleotide sequences recognized by specific DNA-binding proteins (transcription factors) that help initiate transcription.

In eukaryotes, RNA polymerases cannot recognize the promoter without the help os trnascription factors.

Transcription factors + Dna-binding proteins that bind to specific DNA mnucleotide sequences at the promoter and that help RNA polymeraase recognize and bind to the promoter region, so that transcription can begin.

· RNA polymerase II, the ensyme that synthesizes RNA in eukaryotes, usually cannot recognize a promoter unless a spec ifc transcription factore nids to a region on the promoter called a TATA box.

TATA box = A short nucleotide sequence at th prmooter that is rich in thymine (T) and adenine (A) and tha tis ocated anout 25 nucleotides apstream form the initiation site.

· RNA polymerase II recognizes the complex betweent eh bund TATA transcription factor and the DNA binding site.

· Once RNA polymerase recognizes and attaches to the promoter reion, it probably associate with other transcriotio factors before RNA synthesis begins.

When actie RNA polymerase binds to a promoter, the enzyme sepertes the two DNA strands at the initiation site, and transcription beings.

B. Elongation of the RNA strans

on e transcription begins, RNA polymerase II moves along DNA and performs two primary function:

1. It untwists and opes a short segment of DNA exposing about ten nucleotide bases; one of the exposed DNA strands is the template for base-pairig with RNA nucleotides.

2. It links incoming RNA nucleoties to the 2'end of the elongated strandl thysm,RNA grows one nucleotide to the 3' end of hte longating strand; thus, RNA grows one nucloeotide at a time inthe 5' to 3' direction.

During transcription ,. mRNA grows about 30-60 nucleotides per second. As the mRNA strand elongates;

· It peels away from its DNA template

· The nontemplates strand of DNA re-forms a DNA-DNA double helix by pairing with thte template strand.

Following in series, several molecules of RNA polymerase II can simultaneously transcrive the same gene.

· Cells can thus produce3 particular proteins in larege amounts.

· TRh growing RNA strands hang free fromeach polymerase. The lencth of each strand varies and reglects how far the enzyme has traveled from the inititation site on template DNA.

C> Termination of Transcripton

transcription proceeds until RNA polymerse reaches a termination site ont eh DNA>

Terminator sequence + DNA squemce that signals RNA polymerase to stop transcription and to release the RNA molecule and DNA template.

· Additional proteins may cooperate with RNA polymerase in termination.

· In eukaryotes, the most common terminator sequence is AATAAAA.

Prokaryotic mRNA is ready for translation as soon as it leaves the DNA template. Eukaryotic mRNA , however must e processed before it leaces the nucleus and become functional.

V. Translation is the RNA-directed synthesis of a polypeptide: a closer look

During translation, proteisn are sunthesized according to a gentic mesagte of sequentail codons alond mRNA.

· Tranfer RNA (tRNA) is the interpreter between the two forms of information - base sequence in mRNA and amino acid sequence in polypeptides.

· tRNA aligns the appropriate amino acids to form a new polypeptide. To perform this fnction, tRNa must

Tranfwer amin oacids formt he sytoplasms amino acid pool to a ribosome

recognize the correcrt codons in mRNA.

Molecules of tRNA are specific for only one particular amino acid. Each type of tRNA associates a distict mRNA codon with one of the 20 amin acids used to make proteisn.

· One end os a tRNA molecule attaches toa specific amino acid.

· The other end attaches to an mRNA codon by base pairing with its anticodon.

Anticodon = A nucleotide triplet in tRNA that base pairs with a complementary nucleotide triplet (codon) in mRNA.

tRNA s decode the genetic message, codon by codon. Fo example,

· THe mRNA codon UUU is translated as the amino aci phenylanine

· The tRNA that tranfers phenylalanine to the ribosome has an anticodon of AAA.

· When the codon UUU is presented for translation,.phenya;amome wo;; ne addede to the growing polypeptide.

· As tRNAs deposit amin oacids int he correct order, ribosomal enzyems link them into a chain.

A. The structure and Function of Transfer RNA

All types of eukaryoptic RNA, including tRNA, are transcribed form tempalte DNA located wit6hin the nucleus.

· tRNA must travel fromteh nucleus to the cytoplasm , where translation ovccurs.

· Once in the cytoplasm , each tRNA molecul;e can be used repeatedly.

The ability of tRNA to cary specific amino acids and to recognize the corrct codons depends pon its structure; its form fits funciton.

· tRNA is a single-stranded RNA only anout 80 nucleotides long.

· The strand is folded , forming seeral double standed regions where shrot base sequenceces hydrogen bond with other complementary baase sequences.

· A single-plane view reveals a clover leaf shape.

The three-dimensinal structore is roughly L-shaped

· A loop protrudes at one end os the L and has a specialized sequence of three bases called the anticodon.

· At th other end of the Lprotrudes the 3' rnd of the tRNA molecule - the attachment site for an amin acid.

There are only about 45 distinct types of tRna. however, this is enough to translate the 64 codons, since some tRNAs recognize two or three mRNA codons specifying the same amino acid.

· This ispossible because the base-pairin rules are relaxed between the thired base of an mRNA codon and the corresponding base of a tRNA anticodon.

· This exception to the base-pairing rul;e is called wobbled.

Wobble + The abnility of one tRNA to recognize two or three dfferent mRNA codons; occurs when the third ase (5' end ) of the tRNA anticodon has some play or wobble , so that it can hydrogen bond with more than one kind of base in the third position (3' end) pf tj cdpm

· Fr example, the base U in the wobble position of a rRNA anticodon can pair with either A or G in the third position of an mRNA codon.

· Some tRNAs contain a modified base called inosine (I) , which is in the anticodon

· s wobble position and can base pair with U, C or A in the third position of an mRNA codon.

· Thus, a single tRNA with the anticodon CCI will recognize three mRNA codons: GGU, GGC, or GGA - all of which code for glycine.

B. Aminoacyl-tRna Synthetases

The correct lnkage between tRNA and its designated amino acid must occur before the anticodon pairs with its complementary mRNA codon. this process of correctly pairing a tRNA with its appropriate amin oacid s catalyze by an aminoacyl-tRNA synthetase.

Aminoacyl-tRNA synthetase = A type of enzye that catalyzes th attachment of an amino acid to its tRNA

· Each of the 20 amino acids has a specific aminoacyl-tRNA synthetase.

· In and endergonic reaction driven by the hydrolyiss os ATP, the proper synthetase attaches an amino acid to its tRNA in two stpes:

1. Activation aof thamino acid wtih AMP. The syntheetase 's actie site binds the amino acid and ATP; the ATP loses two phosphate groups and attaches to the amino acid a AMP (adenosine monophosphate).

2. Attacmetn of the amino aid to tRNA. The appropriate tRNA covalently bonds to the amino acid, despalcing AMP from the enzymes active site.

· The aminoacyl-tRNA complex releases from the enzyme and transfers itts amino acid to a growing polypeptide on the ribosome.

B. Ribosomes

Ribosomes coordinate the pairing of tRNA anitcodons to mRNA codons.

· Ribosomes have two subunits (small and large) which are sperated when not involvede in protein synthesis.

· Ribosomes are composed of aobut 60% ribosomal RNA (rRNA) and 40% protein.

The large ad small subunits of eukaryotic ribosomes are:

· Constructed in the nucleoous.

· Dispatched through nuclear pores to the cytopplasm.

· Once in the cytoplasm, are assembled into funct6onal ribosomes only when attached to an mRNA.

A. The structure and Function of Transfer RNA

All types of eukaryotic RNA, including tRNA, are transcribed from template DNA locaed wihtin the nucleus.

· TRNA must travel from the nucleus to the cytoplas, where translation occurs.

· Once in the cytoplasm, each tRNA molecule can be used repeatedly.

The ability of tRNA to carry specific amino acids and to recognixe the correct codons depends upon iots structure; its form fits function.

· TRNA is a single-stranded RNA only about 8 nucleotides long.

· The strand is folded, forming several double-stranded regions where short base sequences hydrogen bond with other complementar6y base sequences.

· A single-plane view reveals a clovr leaf shape.

The three-dimensional structure is roughly L-shaped

· A loop protrudes at one end of the L and has a specialized sequence of three bases called the anitcodon.

· At the other end of the Lprotrudes the 3’ end of the tRNA molecule - the attachment site for an amino acid.

There are only about 45 distinct types of tRNAa. However, this is enough to translate the 64 codons, since some tRNA recognize two or three mRNA codons specifying the same amino acid.

· This is possible because the base-pairing rulees are relaxed betweenthe third base of an mRNA codon and the corresponding base of a tRNA anticodon.

· This exception to the base-pairing rule is called wobble.

Wobble = The ability of one tRNA to recognize two or three different mRNA codons; occurs when the third base (5’ end) of the tRNA anticodon has some play or wobble, so that it can hydrogen bond with more than one kind of base in the third position (3’end) of the codon.

· For example, the base U in the wobble position of a tRNA anticodon can pair with either A or G in the third position of an mRNA codon.

· Some tRNAs contains a modified base called inosine (I) , which is in the anitcodon’s wobble position and can base pair with U, C or A in the third position of an mRNA codon.

· Thus, a single tRNA with the anitcodon CCI will recognize three mRNA codons: GGU, GGC or GGA – all of which code for glycine.

A. Aminoacyl - tRNA Synthetases

The correct linkage between tRNA and its designated amino acid must occur before the anticodon pairs with its complementary mRNA codon. This process of correctly pairing a tRNA with its appropriate amino acid is catalyzed by an aminoacyl-tRNA synthetase.

Aminoacyl-tRNA synthetas = A type of enzyme that catalyzes the attachment of an amino acid to its tRNA

· Each of the 20 amino acids has a specific aminoacyl-tRNA synthetaase.

· In qan endergonic reaction driven bby the hydrolysis of ATP, the proper syntheatse attaaches an amino acid to its tRNA in two steps:

1. Activation of the amino acid with AMP. The synthetase’s tRNA covalently bonds to the amino acid, displacing AMP from the enzyme’s active site.

· The aminoacyl-tRNA complex releases from the enzyme and transfers its amino acid to a growing polypeptide on the ribosome.

A. Ribosomes

Ribosomes coordinate the pairing of tRNAAA anticodons to mRNA codons.

· Ribosomes have two subunits (small and large) which are sperated when not involved in protein synthesis.

· Ribosomes are compposed of about 60% ribosomal RNA (rRNA) and 40% protein.

The large and small subunits of eukaryotic ribosomes are:

· Constructed in the nucleolus.

· Dispatched through nuclear pores to the cytoplasm.

· Once in the cytoplasm, ar assembled into functional ribosomes only when attached to an mRNA.

Compared to eukaryotic ribosomes, prokaryotic ribosomes are smaller and have a different molecular composition.

· Selection of effective drug therapies against bacterial ppathogens capitallizes on this difference.

· For example, the antibiotics tetracycline and streptomycin can be used to combat bacterial infections, because they inhibit bacterial protein synthesis without tafecting the ribosomes of the eukaryotic host.

In addition to an mRNA bidnign site, each ribosomes has two tRNA binding sites (P and A).

· The P site holds the tRNA cccarrying the growing polypeptide chain.

· Tha A site holds the tRNA carrying the next amino acid to be added.

As the ribosome holds the tRNA and mRNA molecules together, enzymees transfer the new amino acid from its tRNA to the carboxyl end of the growing polypeptide.

A. Building a Polypeptide

The building of a polypeptide , or translation, occurs in three stages: 1) initiation, 2) elongation, and 3) termination.

· All three stages requrie enzymes and other protein factors.

· Initiation and elongation also requrie energy provided by GTP (a molecule closely related to ATP)

1. Initiation

Inititation must brign together the mRNA, the first amino acid attached to its tRNA, and the two ribosomal subunits.

An initiation complex is Assembled as mRNA and a special tRNA binds to a small ribosomal subunit.

· In eukaryotes, the small ribosomal subutnit binds fist to an initiator tRNA with the anticodon UAC. This tRNA carries methionine, the first amino acid to be added.

· The small ribosomal subunit next binds to the 5’ end of mRNA. MRNA has a specific ribosome-recognition sequence at the 5’ upstream end that base pairs with a complementary sequence on rRNA.

· With help from the small ribosomal subunit, bound initiator tRNA finds and base pairs with the initiation or start codon on mRNA. This start codon, AUG, marks the place where translation will begin and is located just dowstream from the ribosome-recognition sequence.

· Assembly of the initiation complex – small ribosomal subunit, initiator tRNA and mRNA – requires:

Þ Protein initiation factors that are bound to the small ribosomal subunit.

Þ One GTP molecule that probably staballiszes the bindings of initiation factors and upon hydrolysis drives the attachment of the large ribosomal subunit.

In the second step, a large ribosomal subunit binds to the small one to form a funcitonal ribosome.

§ Initiation factors attached to the smlall ribosomal subunit are released, allowing the large subunit to bind with the small subunit.

§ The initiator tRNA fits into the TP site on the ribosome.

§ The vaant A site is ready for the next aminoacyl- tRNA.

1. Elongation

Several proteins called elongation factors take pratr in this three-step cycle which adds amino acids one by one to the initial amino acid.

a. Codon recognition. The mRNA codon in the A site of the ribosomes forms hydrogen bonds with anitcodon of an entering tRNA carrying the next amin acid in the cahin.

§ AN elongation factor directs tRNA into th A site.

§ Hydrolysis of GTP provides energy for this ste.

a. peptide bond formation. An enzyme, peptidyl transterase, catalyzes the formation of a paptide bodn between the polypeptide in the P site and the new amino acid in the A site.

§ Peptidyl transferase is part of the large ribosomal subunit and consists of ribosomal proteins and rRNA.

§ The polypeptide seperattes from its tRNA and is tranferred to the new amino acid carried by the tRNA in the A site.

a. Translocation. The tRNA in th P site releases form the ribosome, and the tRNA in the A site is translocated to the P site.

§ During this process, the codon and anticodon remain bonded, so the mRNAAAa and th tRNA move as a unit, bringing the next codon to be translated into the A site.

§ The mRNA is moved through the ribosome only in the 5’ to 3’ direction.

§ GTP hydrolysis prvides energy for each translocation step.

1. T3ermination

Each iteration of the elongation cycle takes about 60 milliseconds and is repeated intil synthesis is complete and a termiantio codon reaches the ribosome’s A site.

Termination codon (stop codon) = Base triplet (codon) on mRNA that signals the end of translation.

§ Stop codons are Uaa, uag AND uga.

§ Stop codons do not code for amino acids.

When a stop codon reaches the ribosome’s A site, a protein release factor binds to the codon and initiates the following sequence of events;

§ Peptidyl tranferase hydrolyzes the bond betweent th ecompleted polypeptide bnd the tRNA in the P ite.

§ This frees the polypeptide and tRNA, so the can bboth release from the ribosome.

The two ribosomal subunits disociate form mRNA and separate back into a small and a large subunit.

A. Polyribosomes

Single ribosomes can make average-sized polypeptides in less than a minute; usually, however, clusters of ribosomes simultaneously translate an mRNA.

Polyribosome= A cluster of ribosome simultaneously translatingan mRNA molecule.

· Oncea ribosome passes the initiation codon, a second ribosome can attach to the mRNA.

· Several ribosomes may translate an mRNA at once, makind many copies of a polypeptide.

A. From Polypeptide to Funcitonal Protein

The biological activity of proteins depends upon a precise folding of the polypeptide chain into a native three-dimensioal conformation.

· Genes determine primary structure, the linear sequence of amino acids.

· Primary structure determines how a polypeptide chain will spontaneously coil and fold to form a three-dimensional molecule with secondary and tertiatary structure.

Some proteins must undego post-translational modification before they become fully functional in the cell.

· Sugars, lipids, phosphate groups , or other additives may be attached to some amino acids.

· One ormore amino acids may be enzymatically cleaved form the leading ( amino) aend of the polypeptide chain.

· Single polypeptide chains may be divided into two or more pieces (e.g. insulin).

· Two or more polypeptides may join as subunits of a protein that has quarternary structure.

VI. Some polypeptid4es have signal sequences that target them to specific destinations in the cell

Eukaryotic ribosomes function either free in the cytosol or bound to endomembranes.

· Bound and free ribosomes are structurally identical and interchangeable.

· Most proteins made by free ribosomes will function in the cytosol.

· Attached to the outside of the endoplamic reticulum, bound ribosomes generally make proteins that are:

Þ Destined for membrane inclusion in endomembrane system (e.g. nuclear envelope , ER< Golgi, lysosomes, vacuoles and plasma membrane).

Þ Secretory proteins destined for export.

There is only one type of ribosomes, and syntheis of all proteins begins in the cytosol. What determines whether a ribosome will be free in the cytosol or attached to rouigh ER?

Þ Messenger RNA for secretory proteins codes for an initial signal sequence of 16-20 hydrophobic amino acids at the amino end of the forming polypeptide.

Þ When a ribosome begins to synthesize a protein with a signal sequence, it moves to the ER membrane by a mechanism that involves two other components.

o Signal recognition particle (SRP). This protein complex moves between the ER membrane and the cytosol. It is an adaptor that atttaches both to the signal sequence of a growing polypeptide and toa receptor protein in the ER membrane (SRP receptor).

o SRP receptor. This receptor protein is built into the ER membrane. The signal recognition particle docks with the receptor , and the ribosomes thus becomes bound to the ER membrane.

Þ Thr ibosome continues protein synthesis and the leading end of the new polypepide (N-terminus) threase into the cistenal space.

Þ The signal sequence is remobed by an enzyme.

Þ Newly formed polypeptide is released from the ribosome and folds into its native conformation.

Þ IF an mRNA does not code for a signal sequence, the ribosome remains free and synthesizes its protein int eh cytosol.

Different signal sequences may also dispatch proteins to specific sites other than the ER> For example, newly formed proteins may be targeted for mitochondria or cloroplasts.

VII. Comparing protein synthesis in prokaryotes and eukaryotes: a review

Transcription and translation are similar in prokaryotes and eukaryotes, but protein synthsis is organizes differently within their cells.

Þ Prokaryotes lack nuclei, so transcription is not segregated from tanslation; consequently, translation may begin as soon as the 5’ end of mRNA peels away from templte DNA, even before transcription is complete.

Þ The significance of a eukaryotic cell’s compartmental organization is that transcription and translation are segregated by the nuclear envelope. This allows mRNA to be modified before it moves formthe nucleus to the cytoplasm. Such RNA processing occurs only in eukaryotes..

VIII. Eukaryotic cells modufy6 RNA after transcription

Before eukaryotic mRNA is exported from the nucleus, it is processed in two ways: a) both ends are covalently altered and b) intervening sequences are removed and the remainder spliced together.

A. Alternation of mRNA Ends

During mRNA processing, both the 5’ ends are covalently modified.

5’ cap= Modified guanine nucleotide (guanosne triphosphate) that added to the 5’ 4end of mRNA shortly after transcription begins, has two important functions:

Þ Protects the growing mRNA from degradation by hydrolytic enzymes.

Þ Helps small ribosomal subunits recognize the attachment site on mRNA’s 5’ end. A leader segment of mRNA may also be part of the ribosome recognition signal.

Leader sequence = Noncoding (untranslated) sequence of mRNA from the 5’ end to the start codon.

The 3’ end, which is transcribed last, is modified by enzymatic addition of a poly-A tail, before the mRNA exits the nucleus.

Poly-A tail = Sequence of about 200 adenine nucleotides added to the 3’ end of mRNA before it exits the nucleus.

Þ May inhibit degredation of mRNA in the cytoplasm.

Þ May regulate protein synthesis by facilitating mRNA’s export form the nucleus to the cytoplasm.

Þ Is nto attached directly to the stop codon, but to an utnraslated trailer segment of mRNA.

Trailer sequence = Noncoding (untranslated) sequence of mRNA from the stop codon to the poly-A tail.

A. Split Genes and RNA Splicing

The original RNA trsanscript accurately reflects the complementary base sequence of the gene in template DNA; however, it is much longer than the mRNA that functions in the cytoplasm.

Þ The original transcript, or precursor mRNA , is heterogenous nuclear RNA.

Heterogenous nuclear RNA (hnRNA) = Pool of RNA in the nucleus tha contains molecules of widely varied sizes; includes primary mRNA transcripts or precursor mRNA (pre-mRNA).

Þ After RNA processing, only a small portion of the original transcript (pre-mRNA) leaves the nucleus as mRNA.

Genes that code for proteins in eukaryotes are not continuous sequences.

Þ Coding sequences of a gene are interrupted by noncoding segments of DNA called intervening sequences, or introns.

· Introns = Noncoding sequences in DNA that interven between coding sequences (exons); are initially transcribed, but not translated, because they are exercised from the transcript before mature RNA leaves the nucleus.

· Coding sequences of a gene are called exons, because theyare eventualy expressed 9translated into protein).

Exons = Coding sequences of a gene that are transcribed and expressed.

· IN 1977, Richard Roberts and Phi9lip Sharp indeendently found evidence for “split genes”; they won the 1993 Nobel Prize for discovery.

Introsn and exons are both transcribed to form hnRNA, but the introns are subsequently removed and the remaining exons linked together during the process of RNA splicing.

RNA splicing = RNA processing that removes introns and joins exons from eukaryotic hnRNA; produces mature mRNA that will move into the ytoplasm form the nucleus.

· Enxymes excise introns and splice together exons to form an mRNA with a continuous coding sequence

· RNA splicing also occurs during post—transcriptional processing of tRNA and rRNA.

Though there is much lft to be discovered, some details of RNA splicing are now known.

· Each end of an int5ron has short bondary sequences that accurately signal the RNA splicing sites.

· Small nuclear ribonuceoroteins (snRNP’s), play a key role in RNA splicing.

Small nuclear ribonucleoproteins ( snRNPs) = Complexes of proteins and small nuclear RNAs that are found only in the nucles; some particpate in RNA splicing. ( SnRNPs is pronounced “snurps”.)

· These small nuclear particles are composed of:

1. Small nuclear RNA (snRNA). This small RNA molecule has less than 300 nucleotides - much shoreter than mRNA.

2. Protein. Each snRNP has seven or more proteins.

· There are various tpes of snRNP’s with different functions; those incolced in RNA splicing are pa5rt of a larger, more complex assembly called a spliceosome.

Spliceosome = A large molecular complex that catalyzes RNA splicing reactions; composed of small nuclear ribonucleoproteins ( snRNPs) and other proteins.

· As the spliceosome is assembled, one type of snRNP base pairs with a complementary sequence at the 5’ end of the intron.

· The spliceosome precisely cuts the RNA transcript at specific splic sites at either end of the intron, which is excised as alariat-shaped loop.

· The intron is released and the adjacent exons ar immediately spliced together by the spliceosome.

C. Ribozymes

Other kinds of RNA transcripts, such as tRNA and rRNA, are spliced differently,; however, as with mRNA splicing , RNA is often involved in catalyzing the reactions.

Ribozymes = RNA molecules that can catalyze reactions by breaking and forming covalent bonds; are called ribozymes to emphasize their enzymelike catalytic activity.

· Ribozymes were first discovered in Tetralymena , a ciliated protozoan that has self-splicing rRNA. That is, intron rRNA catalyzes its own splicing, completely without proteins or extra RNA molecules.

· Since RNA is acting as an enzyme, it can no longer be said that “All enzymes are proteins”.

· It has since been discovered that rRNA also functions aas an 3enzyme during translation.

Three-dimensional conformations vary among the types of RNA.. These differences in shape give RNA its ability to perform a variety of functions, such as:

1. Information carrier. Messenger RNA (mRNA) carries genetic information from DNA to ribosomes; this genetic message specifies a protein’s primary structure.

2. Adapter molecule . Transfer RNA (tRNA) acts as an adapter in protein synthesis by translating information form one form (mRNA nucleotide sequence) into another (protein amino acid sequence).

3. Catalyst and structural molecule. During translation, ribosomal RNA ( rRNA) plays structural and probably enzymatic roles in ribosomes. Small nuclear RNA (snRNA) in snRNP particle also plays structural and enzymatic roles within spliceosomes that catalyze RNA splicing reactions.

4. Viral genomes. Some viruses use RNA as their genetic material.

D. The functional and Evolutionary Importance of Introns

What are the biological functions of introns and gene splicing?

Introns may play a regulatory role in their cell.

· Intron DNA sequences may control gene activity.

· The splicing process itself may regulate the export of mRNA to the cytoplasm.

Introns may allow a single gene to direct the synthesis of different proteins.

· This can occur if the same RNA transcript is processed differently among various cell types in the same organism.

· For example, all introns may be removed from a particular transcript in one case; but in another, one or more of the introns may be left in place to be translated. Thus, the resulting proteins in each case would be different.

Introns play an important role in the evolution of protein diversity; they increase the probability that recombination of exons will occur between alleles.

· In split genes, coding sequences can be separated by long distances, so they have higher recombination frequencies than continuously coded genes without introns.

· Exons of a “split gene” may code for different domains of a protein that have specific functions, such sd, an enzyme’s active site or a protein’s binding site.

Protein domains = Continuos polypeptide sequences that are structural and functional units in proteins with a modular architecture.

· Genetic recombination can occur in just one exon resulting in the synthesis of a novel protein with only one altered domain.

IX. A point mutation can affect the function of a protein

Knowing how genes are translated into proteins, scientists can give a molecualr description of heritable changes that occur in organisms.

Mutation = A permanent change in DNA that can involve large chromosomal regions or a single nucleotide pair.

Point mutation = A mutation limited to about one or two nucleotides in a single gene.

A. Types of Point Mutations

There are two categoreis of point mutations: 1) base=pair substitutions and 2) base-pair insertions or deletions.

1. Substitutions

Base-pair substitution = The replacement of one base pair with another; occurs when a nucleotide and its partner from the complementary DNA strand are replaced with another pair of nucleotides according to base-pairing rules.

Depending on how base-pair substitutions are translated, they can result in little or no change in the protein encoded by the mutated gene.

· Redundancy in the genetic code is why some substitution mutations have no effect. A base pair change may simply transform one codon into another that codes for the same amino acid.

· Even if the substitutions alters an amino acid, the new amino acid may have similar properties to the one it replaces, or it may be in a part of the protein where the exact amino acid sequences is not essential to its activity.

Some base-pair substitutions result in readily detectable changes in proteins.

· Alteration of a single amino acid in a crucial area of a protein will significantly alter protein activity.

· On rare occasions, such a mutation will produce a protein that is improved or has capabilities that enhance success of th mutant organism and its descendants.

· More often, such mutations produce a lesss active or inactive protein that impairs celll function.

Base-pair substitutions are usualy missense mutations or nonsense mutations.

Missense mutation = Base-pair substitution that alters an amino acid codon (sense codon) to a new codon that codes for a different amino acid.

· Altered codons make sense ( are translated), but not necessarily that originally intended.

· Base-pair substitutions are usually missense mutations.

Nosense mutation = Base-pair substituion that changes an amino acid codon (sense codon) to a chain termination codon, or vice versa.

· Nonsense mutations can result in premature termination of translation and the production of a shorter than normal polypeptide.

· Nearly all nonsense mutations lead to nonfunctional proteins.

2. Insertions of Deletions

Base-pair insertions or deletions usually have a greater negative effect on proteins than substitutions.

Base-pair insertion = the insertion of one or more nucleotide pairs into a gene.

Ase-pair deletion = The deletion of one or more nucleotide pairs from a gene.

Because mRNA is read as a series of triplets during translation, insertion or deletion of nucleotides ma alter the reading frame (triplet grouping) of the genetic message. This type of frameshift mutation will occur whenever the number of nucleotides inserted of deleted is not 3 or a multiple of 3.

Frameshift mutation = A baswe-pair insertion or deletion that causes a shift in the reading frame, so that codons beyond the mutation will be the wrong grouping of triplets and will specify the wrong amin oacids.

· A fremshift mutation causes the nucleotides following the insertion or deletion to be improperly grouped into codons.

· This results in extensive missense, which will sooner or later end in nonsesnse (premature termination).

· Frameshift will produce a nonfuncitonal protein unless the insertion or deletion is very nar the end of the gene.

B. Mutagens

Mutagenesis = The creation of mutations.

· Mutations can occur as errors in DNA replication, repair or recombination that result in base-pair substitutions, insertions or deletions.

· Mutagenesis may be a naturally occuring event causing spontaneous mutations, of mutations may be caused by exposure to mutagens.

Mutagen = Physical or chemical agents that interact with DNA to cause mutation.

· Radiation is the most common physical mutagen in nature and has been usd in laboratory to induce mutations.

· Several categories of chemical mutagens are kown inclduing base analogues, which are chemicals that mimic normal DNA bases, bus base pair incorrectly.

· The Ames test, developed by Bruce Ames, I oe of the most widely used test for measuring the mutagenic strangth of various chemicals. Since most mutagens are carcinogenic, this test is also used to screen for chemical carcinogens.