Chapter 10

Molecular Biology of the Gene

Information transfer is from DNA RNA protein

ReplicationWhat is it?Where does it occur?

REPLICATION

Copying DNA for division

In the nucleus

Information transfer is from DNA RNA protein

TranscriptionWhat is it?Where does it occur?

Making mRNA from DNA

In the nucleus

Information transfer is from DNA RNA protein

TranslationWhat is it?Where does it occur? In the cytoplasm, at a ribosome

Converting mRNA into a protein

2. DNA as source of genetic information

a. Hershey-Chase experiment showed DNA rather than protein to be the genetic material passed on from one generation to the next

DNADNA

DNADNA

DNADNA

DNADNA

DNADNA

DNADNA

2. DNA as source of genetic information

b. additional evidence – cell doubles DNA prior to mitosis, and then splits the DNA evenly among daughter cells

Watson and Crick

3. Molecular structure of DNA

a. Watson and Crick described the three dimensional structure of DNA one year after Hershey and Chase identified DNA as the genetic material

3. Molecular structure of DNA

b. DNA, along with RNA, are nucleic acids which are composed of nucleotides

c. Nucleotides consist of a sugar (ribose or deoxyribose), a nitrogenous base (A, G, C, T, or U), and a phosphate group

3. Molecular structure of DNA

d. Structure of single DNA strand 1. sugar-phosphate backbone 2. bases covalently attached to sugar

and ‘hang off’ the side

3. Molecular structure of DNA

e. double helical structure 1. double stranded 2. arranged in helix

3. Molecular structure of DNA

3. hydrogen bonds between nitrogenous bases hold strands together (remember, hydrogen bonds are weak chemical bonds)

3. Molecular structure of DNA

4. the two strands of DNA run “anti-parallel”; i.e., one strand runs in 5’-3’ direction while the other runs in the 3’-5’ direction The primed numbers refer to the C of the sugar. The bases are attached to the 1’ carbon and the phosphate groups are attached at the 5’ sugars. Nucleotides form covalent bonds between the 3’ carbon of one and the 5’ carbon of the other nucleotide.

4. DNA replication

a. complementary base pairing governs how new DNA molecules are synthesized using existing DNA as templates (fig 10.4)

1. A with T 2. G with C

4. DNA replication

b. DNA synthesis is semiconservative; i.e., the two strands are separated and each strand is used as a separate template.

4. DNA replication

c. DNA synthesis occurs along each of the separated strands thus resulting in two new double-stranded molecules of DNA

4. DNA replication

d. New nucleotides are added to a growing strand of DNA one at a time, and this energy-requiring reaction is catalyzed by an enzyme, DNA polymerase

4. DNA replication

e. The new strands are synthesized 5’-3’ and anti-parallel with the template strands (10.5)

4. DNA replication

f. The two new strands of DNA are synthesized as the leading and lagging strand

4. DNA replication

g. process of replication 1. the enzyme helicase unwinds the

double stranded DNA, while single stranded binding proteins stabilize the templates

4. DNA replication

2. primase adds RNA primers to the exposed templates because DNA polymerase must add new nucleotides to a 3’ end of an existing nucleotide in an already started strand

5’ 3’

3’ 5’

4. DNA replication

3. DNA polymerase adds one nucleotide at a time in the 5’ – 3’ direction along the leading strand and lagging strand (leading strand is synthesized continuously while the lagging strand is synthesized in Okazaki fragments)

4. DNA replication

4. Another DNA polymerase replaces the RNA primer

5. Ligase seals the Okazaki fragments

1. Overview of protein synthesis

Process = DNA to RNA to protein

1. Overview of protein synthesis

Specific sequences of DNA in genes code for specific sequences of RNA which in turn code for specific sequences of amino acids in proteins

1. Overview of protein synthesis

a. compartmentalization1. transcription in nucleus

2. translation (protein synthesis) in cytoplasm

2. Genetic Code

a. mRNA is read 3 nucleotides at a time; i.e., one amino acid coded for by three nucleotides

2. Genetic Code

b. each set of three nucleotides is referred to as a codon

c. use genetic code of RNA codons to predict amino acid sequence in synthesized peptide

2. Genetic Code

c. use genetic code of RNA codons to predict amino acid sequence in synthesized peptide

Using the Chart

CAU

• The codon CAU codes for His

3. Transcription

a. Initiation- RNA polymerase binds to promoter sequence of DNA, unwinds DNA and starts transcription at start site

3. Transcription

b. Elongation – RNA polymerase makes new strand of RNA in 5’ to 3’ direction; i.e., it adds new nucleotides to the 3’ end of the growing RNA strand, DNA reforms double strand behind polymerase

AUG CAU GUC GAU CAC UAA AGU UUATAC GTA CAG CTA GTG ATT TCA AAT

ATG CAT GTC GAT CAC TAA AGT TTA

ATG CAT GTC GAT CAC TAA AGT TTA

3. Transcription

c. Termination – RNA polymerase reaches a terminator sequence of DNA and polymerase along with the newly synthesized mRNA are released

3. Transcription

d. Eukaryotic RNA is processed in the nucleus before final mRNA is sent to cytoplasm

3. Transcription

e. One gene (DNA) is read at a time by RNA polymerase in eukaryotes (monocystronic)

3. Transcription

f. Multiple genes can be read at a time by RNA polymerase in prokaryotes (polycystronic)

4. Translation

a. synthesis of proteins using RNA as a template

b. catalyzed by ribosomes in the cytoplasm

What Translation Looks Like

4. Translation

c. involves a variety of other players

1. t RNA transfer

2. m RNA messenger

3. r RNA ribosomal

5. tRNA

a. interpreters between nucleic acid language and protein language; i.e., translation

b. single stranded nucleic acid made via transcription just like mRNA

5. tRNA

c. 3’ end of tRNA binds amino acid

d. anticodon sequence of tRNA base pairs with corresponding codon on mRNA; therefore, anitcodon – codon binding determines which amino acid is added to the growing peptide

6. Ribosome (fig 10.12)

a. Catalyze protein synthesis

b. two ribosomal subunits; large and small

6. Ribosome

c. mRNA binding site on small ribosomal subunit

d. two tRNA binding sites known as P and A on large ribosomal subunit

6. Ribosome (fig 10.12)

e. an anticodon of a tRNA binds to the ribosome when its anticodon base pairs with a mRNA codon present in that same binding site

6. Ribosome (fig 10.12)

f. P site holds the tRNA attached to growing peptide

g. A sites holds the tRNA attached to the new (incoming) amino acid

What Translation Looks Like

7. Initiation of translation

a. small ribosomal subunit binds mRNA

b. a special initiator tRNA with anticodon UAC binds to start codon AUG (this tRNA carries amino acid methionine)

7. Initiation of translation

c. large ribosomal subunit binds with small ribosomal subunit placing initiator tRNA in P site and leaving A site empty for the next tRNA to bind

8. Elongation of translation (fig 10.14)

a. an incoming tRNA/amino acid binds to unoccupied A site

b. ribosome catalyzes formation of peptide bond between new amino acid and growing peptide, and the growing peptide is released from the tRNA in the P site

c. tRNA in A site is translocated to P site, moving the mRNA along with it a distance of 3 nucleotides; i.e., one codon

d. the mRNA moves along the ribosome one codon at a time

9. Termination of translation

a. The A site of the ribosome reaches a stop codon (UAA, UAG, or UGA) in the mRNA molecule

b. a releasing factor binds to the stop codon instead of another tRNA molecule

c. Releasing factor catalyzes release of peptide from ribosome

d. Translation assembly falls apart and can be used again

10. Overview of translation (fir 10.15)

a. amino acids polypeptide (protein)

b. mRNA carries the “message” of the genetic code from the nucleus to the cytoplasm

c. tRNA/amino acid complex in cytoplasm

d. ribosome brings tRNA/amino acid to mRNA in a particular order as dictated by mRNA nucleotide sequence

e. ribosomes catalyze binding of amino acids into polypeptide; i.e., formation of peptide bonds

Mutations Mutations are random changes in the DNA

sequence. Gene mutations are relatively small

affecting only one or two genes. Point mutations are caused by

substitutions and usually result in the change of one amino acid, and causing no change about 30% of the time.

Frameshift mutations are usually caused by a deletion. The affect all of the codons that follow the deletion. This will change many of the amino acids in the protein molecule.

Substitution / Point Mutation

AUG CAU GUC GGU CAC UAA AGU UUA

AUG CAU GUC GAU CAU UAA AGU UUA

AUG CAU GUC GAU CAC UAA AGU UUA

AUG CAU GUC GAU CAC GAA AGU UUA

Deletion / Frameshift

AUG CAU GUC GAU CAC UAA AGU UUA

AUG CAU GUC GAU CAC UAA AGU UUA

AUG CAU GUC GUC ACU AAA GUU UAG