Ch. 17: From Gene to Protein

I. Intro:A.Genetic material is in the form of specific sequences of nucleotides along DNA.

How is this sequence translated into a specific trait (Brown eyes, Blue eyes …)?

B. This chapter explores how DNA inherited by an organism leads to specific traits by dictating the synthesis of proteins.

II. The connection between genes and proteins.A. The study of metabolic defects provided

evidence that genes specify proteins.1. In 1909, Archibald Garrod was first to

suggest that genes dictate phenotype.

2. He studied an inherited disease calledalkaptonuria, which causes urine to beblack. He said that the cause of it was due to a person’s inability to make a particular enzyme.

-Individuals with this disease contains alkapton in the urine, which causes the urine to look black. Normal individuals have an enzyme that metabolizes alkapton.

B. How genes control metabolism: One gene = one protein

1. George Beadle and Edward Tatum studied bread mold, Neurospora crassa.

a. They X-rayed the mold and then looked for mutant survivors that differed in their nutritional needs.

-Wild-type mold can survive on minimal medium agar (made up of salts, glucose, and vitamin biotin.-Mutant mold could only survive on complete growth medium, supplemented with 20 amino acids and a few more nutrient.

The mutant mold were unable to synthesize essential molecules froma minimal medium.

-One mutant only needed the addition of amino acid arginine to a minimal medium. Beadle and Tatum concluded that the mutant was defective in the biochemical pathway that synthesizes arginine. The mutant was deficient in an enzyme that synthesizes arginine. Their study provided strong evidence for one gene = one protein hypothesis.

2. Later, Beadle and Tatum’s one gene=one protein was changed to:one gene = one polypeptideWe now know that proteins can bemade up of many polypeptides, each ofwhich have their own gene.

C. Transcription and translation are the twomain processes linking gene to protein.1. Before protein can be made from DNA,

RNA must be made. It is made in a process called transcription.

2.Three differences between DNA and RNA: a. RNA is a single strand

b. RNA has Uracil instead of Thymine c. Sugar in RNA is ribose

3. During transcription, a DNA strand provides a template for the synthesis of a complementary RNA strand.

-The complementary RNA strand made is called mRNA (messenger RNA).

4. During translation, the information contained in the order of nucleotides in mRNA is used to determine the amino acid sequence of a polypeptide.

-A ribosome helps synthesize proteins from the mRNA.

5. Transcription and translation is similarin eukaryotes and prokaryotes.

a. In prokaryotes, transcription and translation can happen simultaneouslybecause there is no nucleus.

b. In eukaryotes, transcription takes place in the nucleus and translationtakes place in the cytoplasm.

-However, before the mRNA leaves the nucleus, it is processed.

6. A summary of protein synthesis:DNA mRNA Proteintranscription translation

D. In the genetic code, nucleotide triplets specify amino acids.

1. The genetic instruction for a polypeptide chain are written in DNA as a series of three-nucleotide words.

Example: AGT = serine 2. During transcription, one DNA strand,

the template strand, provides a template for ordering the sequence of nucleotides in an RNA transcript.

3. The complementary RNA molecule is synthesized according to base-pairing rules, except that uracil is the complement-ary base to adenine.

4. Every 3 mRNAnucleotides is acodon, which codes for a specific amino acidin translation.

5. Translation takes place in a 5’3’ direction.

6. Cracking the genetic code:a. The first codon was deciphered in

1961 by Marshall Nirenberg. He discovered that UUU coded for Phenylalanine.b. By the mid 60’s, the code was

cracked:

PhePheLeuLeu

SerSerSerSer

TyrTyr

STOPSTOP

CysCys

STOPTryp

LeuLeuLeuLeu

ProProProPro

HisHisGlnGln

ArgArgArgArg

IleIleIle

Met

ThrThrThrThr

AsnAsnLysLys

SerSerArgArg

ValValValVal

AlaAlaAlaAla

AspAspGluGlu

GlyGlyGlyGly

U

C

A

G

Firs

t Le

tter

of

Codon

U C A G

Second Letter of Codon

UCAG

UCAG

UCAG

UCAG

Th

ir d Le

tt er o

f Cod

on

c. Each polypeptidestarts withMethionine.AUG is the

start codon.

d. Three codons donot code foran aminoacid. Theyare stop codons, which stops translation.

e. There is redundancy in the genetic code.

Example: GAA and GAG both code for the same amino acid.

f. To extract the correct message fromthe genetic code, there must be aspecific starting point. -A correct reading frame must be

ensured.E. The genetic code must have evolved very

early in the history of life.1. The genetic code is universal, shared by

the simplest bacteria to the most complex plants and animals.Example: CCG codes for proline in all organisms.

2. Since the genetic code is shared by organisms, we can insert human genesin bacteria so they can synthesize certain human proteins. BIOTECHNOLOGY

In this picture, a tobacco plant has a firefly gene implanted in its DNA.

3. Exceptions in the universal genetic code:-Some mitochondrial and chloroplast codons differ.-Paramecium also have some codons that differ.

III. The Synthesis and Processing of RNAA. Transcription: DNA-directed synthesis of

RNA1. mRNA is transcribed from a template

strand of DNA.

2. Before a new mRNA strand can be made,the DNA strand must be separated by theenzyme called RNA Polymerase.

3. RNA polymerase then adds nucleotides only to the 3’ end of the growing polymer.

4. Genes on DNA are read from 3’5’, andmRNA nucleotides are add from 5’3’.

5. Specific sequences of nucleotides along the DNA mark where gene transcription begins and ends.

-RNA Polymerase attaches to the starting point called the promoter.-The gene to be transcribed is called the transcription unit.

-Transcription ends when RNA polymerase reaches the terminator.

6. Bacteria have one type of RNA polymerase.

7. Eukaryotes have 3 types of RNA polymerase (I, II, III) in their nucleus.

a. RNA polymerase II synthesizes mRNA.8. Transcription can be divided into

3 stages: Initiation, Elongation, Termination

a. In initiation in eukaryotes, proteins called transcription factors recognize the promotor region, especially a TATA box, and bind to the promotor.

-After the factors bind to the TATA box, RNA poly- merase binds to the factors, creating the transcription initiation complex.

-RNA polymerase then starts transcription.

-As RNA polymerase moves along the DNA, it untwists the double helix, 10 to 20 bases at time.

-Nucleotides are added at the 3’ end.-A single gene can be transcribed simultaneously by several RNA polymerases. This helps the cell make proteins in large amounts.

b. Elongation continues until after the RNA polymerase transcribes a terminator sequence in the DNA.

c. Termination in prokaryotes takes place right when the RNA polymerasereaches the terminator sequence. Both RNA and DNA are released.

-Termination in eukaryotes takes place when RNA polymerase continues for hundreds of nucleotides past the terminator sequence, AAUAAA. Then at a point about 10 to 35 nucleotides past this sequence, the pre-mRNA is cut from the enzyme.

9. Eukaryotic cells modify RNA after transcription:a. Enzymes modify pre-mRNA before it

enters the cytoplasm.b. At the 5’ end of the pre-mRNA mole-

cule, a modified form of guanine is added. This is the 5’ cap.

-This helps protect mRNA from hydrolytic enzymes.-Its also a signal for ribosomes to attach to the mRNA strand.

c. At the 3’ end, an enzyme adds 50 to 250 adenine nucleotides. This is the poly(A) tail.

-In addition to inhibiting hydrolysis and facilitating ribosome attachment, the poly(A) tail also seems to facilitate the export of mRNA from the nucleus.

d. The mRNA strand also has a non-translated leader (before start codon)and a trailer (after stop codon).

e. RNA Splicing: The removal of a largeportion of RNA.-Most eukaryotic DNA and their RNA transcripts have long noncoding stretches of nucleotides.These are called introns. They lie between coding regions of DNA called exons.-After splicing, the final mRNA product contains the exons and the leader and trailer sequences.

-RNA splicing removes introns and joins exons to create an mRNA molecule with a continuous coding sequence.

-This splicing is accomplished by a spliceosome.A spliceosome consists of a variety of proteins and several small nuclear ribonucleoproteins (snRNPs).Each snRNP is made up of proteins & a small nuclear RNA (snRNA) segment(150 nucleotides long).

-In this process, the snRNA acts as a ribozyme, an RNA molecule that functions as an enzyme.-The discovery of ribozymes rendered obsolete the statement, “All biological catalysts are proteins.”

-One role of introns is to provide Alternative RNA splicing. Alternative RNA splicing gives rise to two or more different polypeptides, depending on which segments are treated as exons.

Different exonscode for differentdomainsof a protein.

-Another role of introns is to increase the opportunity for recombination between two alleles of a gene.

IV. The Synthesis of Protein: TranslationA. Codons on an mRNA molecule is translated

into protein in translation.1. Codons are translated by a molecule

called tRNA (transfer RNA).a. tRNA transfers

amino acids from the cytoplasm to a ribosome.

b. The ribosome adds each amino acid to the growingchain of aminoacids.

c. During translation, tRNA links a mRNA codon with the appropriate amino acid.

d. Each tRNA arriving at the ribosome carries a specific amino acid at one end and has a specific nucleotide triplet, an anticodon, at the other.

e. The anticodon base-pairs with a complementary codon on mRNA.

f. tRNA are reused repeatedly. Each time,they pick up their designated aminoacid. They deposit it at the ribosome and then return to the cytosol to pick up another copy of that amino acid.

g. The anticodons of some tRNAs recognize more than one codon.

This is possible the base pairing between the third base of the codon and anticodon are relaxed (called wobble).

At the wobble position, U on the anticodon can bind with A or G in the third position of a codon.Some tRNA anticodons include a modified form of adenine, inosine, which can hydrogen bond with U, C, or A on the codon.

h. Each amino acid is joined to the tRNAby an enzyme called aminoacyl-tRNA synthetase.

-20 different synthetases match the 20 different amino acids.-The synthetase catalyzes a covalent bond between them, forming aminoacyl-tRNA or activated amino acid.

B. Ribosomes facilitate the specific coupling of the tRNA anticodons with mRNA codons.1. Each ribosome has a small subunit and a

large subunit.

2. Ribosomes are made up of rRNA (ribosomal RNA) and protein.

a. The two subunits join to form a functional ribosome only when attachedto a mRNA strand.

3. Ribosomes between prokaryotes andeukaryotes differ enough that tetracycline, an antibiotic that can paralyze prokaryotic ribosomes.

4. Each ribosome has a binding site for mRNA and three binding sites for tRNA molecules.

a. P site: holds the tRNA carrying the growing polypeptide chain.b. The A site carries the tRNA with the next

amino acid.c. Discharged tRNAs leave the ribosome at

the E site.

C. Translation has three stages:Initiation, Elongation, Termination

-Initiation and elongation require GTP for energy.

1. Initiation: brings together mRNA, a tRNA with the first amino acid, and the two ribosomal subunits.a. First, a small ribosomal subunit binds

with mRNA and a special initiator tRNA, which carries methionine and attaches to the start codon.

b. Initiation factors brought the largesubunit to form the translation initiation complex.

2. Elongation: consists of a series of three step cycles as each amino acid is added to the proceeding one.

a. Codon recognition: An elongation factorassists hydrogen bonding between the mRNA codon under the A site with the corresonding anticodon of tRNA carrying the appropriate amino acid.

-This step requires 2 GTP.

b. Peptide bond formation: an rRNA molecule catalyzes the formation of a peptide bond between the polypeptide in the P site with the new amino acid in the A site.

After a peptide bond forms, the growing amino acid chain is moved from the P site to the tRNA on the A site.

c. Translocation: the ribosome moves the tRNA with the attached polypeptide from the A site to the P site.Now that the A site is available, the next tRNA with the designated aminoacid will attach at the A site.

The tRNA that had been in the P site is moved to the E site and then leaves the ribosome. Translocation requires 1 GTPmolecule. The mRNA is read in a 5’3’direction.

Elongation continues until the poly-Peptide chain is completed.

3. Termination: occurs when one of the three stop codons reaches the A site.

a. A release factor binds to the stop codon and hydrolyzes the bond between the polypeptide and its tRNA in the P site.

D. Typically a single mRNA is used to make many copies of a polypeptide simultaneously.1. Multiple ribosomes, polyribosomes,

may trail along the same mRNA.

2. During or immediately after the poly-peptide is synthesized, the polypeptidestarts to fold into its secondary andtertiary structure.

a. This folding depends on the primarystructure of protein (amino acid sequence).

b. Chaperone proteins can also aid inthe folding of the protein.

c. The polypeptide can undergo otherposttranslational modification: -Addition of sugar, phosphate, lipids-Enzymes may cleave the polypeptide-Two or more polypeptides may join together to form a protein (quaternary structure).

E. Signal peptides target some eukaryotic polypeptides to specific destinations in the cell.

1. Translation in all ribosomes begins in the cytosol, but a polypeptide destined for the endomembrane system or for export has a specific signal peptide region at or near the leading end (about 20 amino acids).

2. A signal recognition particle (SRP) binds to the signal peptide and attaches it and its ribosome to a receptor protein in the ER membrane. The SRP is made up proteins and RNA.

3. After binding, the SRP leaves and protein synthesis continues inside the cisternal space of the ER via a protein pore.

-Proteins that will be embedded in the plasma membrane will remain partially embedded in the ER membrane.-Secretory proteins will be released completely into the cisternal space of the ER.

4. Some signal peptides target mitochondria, chloroplasts, the nucleus, and other organelles that are not part of the endomembrane system.

-These proteins are made in the cytosol.

F. RNAs roles in protein synthesis: an overview

1. The diverse functions of RNA is partly due to its ability to hydrogen bond with other nucleic acids.

G. Comparing protein synthesis in eukaryoteswith prokaryotes: a review

1. Prokayotes can transcribe and translatethe same gene at the same time.

2. In eukaryotes,the nuclearmembrane separates transcription and translationin both space and time.

3. In eukaryotes, RNA is processed before translation can take place.

4. Eukaryotes also have complicated mechanisms for targeting proteins to the appropriate organelle.

H. Mutations can affect protein structureand function.1. Mutations are changes in the genetic

material of a cell (or virus).2. Mutations include: inversions,

translocations, and duplications. 3. Point mutations are a chemical change

in just one base pair.

4. Example of a point mutation: Sickle Cell Anemia

a. This is caused by one mutation of a single base pair in the gene that codes for one of the polypeptides of hemoglobin.

b. A change in a single nucleotide from T to A in the DNA template leads to an abnormal protein.

5. This is a type of point mutation called a base-pair substitution.

6. Some base pair substitutions have little to no effect.

a. Silent mutation: When there is a mutation, but the new codon still codes for the same amino acid.

7. Some base pair substitutions havenoticeable effects. They can be detrimental, with the occasionalimproved protein.a. Missense mutation: A base pair

substitution mutation thatcauses a change in amino acid.

b. Nonsense mutation: A base pairsubstitution mutation that causesthe codon to become a stop codon.

8. Insertions and deletions are additionsor losses of nucleotide pairs.a. These have more disastrous effects

than a substitution mutation.

b. They can cause a frameshift mutation.

c. A frameshift mutation can regroup codons improperly.

I. Mutations can occur spontaneously duringDNA repair, DNA replication or DNA recombination.

1. Mutations can also be caused bychemicals or physical agents.

a. Physical: UV and X-ray radiation.b. Chemical: Base analogues that can

be substituted into DNA, but pair upincorrectly.

J. Protein Synthesis: a review

1. Transcription,RNA processing, and translation are the processes that link DNA sequences to the synthesis of a specific polypeptide chain.