MOLECULAR BIOLOGY: REPLICATION, TRANSCIPTION,

AND TRANSLATIONHonors Biology

2012

IMPORTANT EXPERIMENTS

• Frederick Griffith

• Described a “transforming factor” that could be transferred into a bacterial cell

• Process is called transformation

IMPORTANT EXPERIMENTS

• Hershey-Chase

• Used bacteriophages to show that DNA is the genetic material

• Phages were labeled with radioactive sulfur (labels proteins) and radioactive phosphorus (labels DNA)

• Sulfur-labeled protein stayed outside the cell

• Phosphorus-labeled DNA was inside the cell

Head

Tail fiber

DNA

Tail

Batch 1 Radioactive protein

Bacterium

Radioactive protein

DNA

Phage

Pellet

Radioactive DNA Batch 2

Radioactive DNA

Empty protein shell

Phage DNA

Centrifuge

Radioactivity in liquid

Measure the radioactivity in the pellet and the liquid.

4 Centrifuge the mixture so bacteria form a pellet at the bottom of the test tube.

3 Agitate in a blender to separate phages outside the bacteria from the cells and their contents.

2 Mix radioactively labeled phages with bacteria. The phages infect the bacterial cells.

1

Pellet

Centrifuge Radioactivity in pellet

1

2

3

NUCLEOTIDES • Monomer of DNA and RNA is

called a nucleotide

• Contains three parts: nitrogenous base, 5-carbon sugar, and phosphate group

• DNA and RNA are called polynucleotides

• Composed of a sugar-phosphate backbone

• Nitrogenous bases point inward

• Four DNA bases: thymine, cytosine, adenine, and guanine

Sugar-phosphate backbone

DNA nucleotide

Phosphate group

Nitrogenous base Sugar

DNA polynucleotide

DNA nucleotide

Sugar (deoxyribose)

Thymine (T)

Nitrogenous base (A, G, C, or T)

Phosphate group

Pyrimidines

Guanine (G) Adenine (A) Cytosine (C) Thymine (T)

Purines

WATSON AND CRICK• Credited with

discovering the structure of DNA

• Really took ideas from others and were the first to publish

• Took X-ray crystallography data from Rosalind Franklin without her knowledge

DNA STRUCTURE• Composed of two

polynucleotide chains joined together by hydrogen bonds between bases

• A pairs with T, forming two hydrogen bonds

• G pairs with C, forming three hydrogen bonds

• Twisted as a helical shape

Hydrogen bond

Base pair

Partial chemical structure Computer model Ribbon model

Twist

4

5

6

DNA REPLICATION

• Semiconservative model

• Two DNA strands separate

• Each strand is used as a pattern to produce a complimentary strand

• Each new DNA helix has one old strand and one new strand

Parental molecule of DNA

Nucleotides

Both parental strands serve as templates

Two identical daughter

molecules of DNA

DNA REPLICATION• Replication occurs in the 5’ → 3’

direction

• Leading strand = continuous replication

• Lagging strand = segmented replication

• Enzymes important for replication

• DNA helicase binds to unwind the DNA

• DNA polymerase adds new base pairs

• DNA ligase joins fragments together

Origin of replication Parental strand Daughter strand

Bubble

Two daughter DNA molecules

3! end 5! end

3! end 5! end

3!

5!

2!

4! 1! 3!

5!

2!

4! 1!

P

P

P

P

P

P

P

P

Parental DNA 3! 5!

DNA polymerase molecule

DNA ligase

3! 5!

Overall direction of replication

Daughter strand synthesized continuously

3! 5!

3! 5!

Daughter strand synthesized in pieces

FROM DNA TO PROTEIN

• Gene sequence of DNA directs for the synthesis of a protein

• DNA is transcribed into RNA

• RNA is translated into protein

Cytoplasm

Nucleus

DNA

Transcription

RNA

Translation

Protein

Polypeptide

Translation

Transcription

DNA strand

Codon

Amino acid

RNA

7

8

9

GENETIC CODE• Codons - three nucleotide

sequences that correspond to a particular amino acid

• 61 codons code for an amino acid (more than one codon can code for an amino acid)

• AUG - start codon (codes for the amino acid methionine)

• Three stop codons (UAA, UAG, and UGA)

Firs

t bas

e

Thir

d ba

se

Second base

TRANSCRIPTION• DNA strands separate

• One strand is used as a patter to produce RNA

• Important change: In RNA thymine (T) is not used; uracil (U) is used instead.

• RNA polymerase is the enzyme that complete transcription

• Three stages:

• Initiation: RNA polymerase binds to a promoter to unwind the helix

• Elongation: RNA nucleotides are added to the chain

• Termination: RNA polymerase reaches a terminator sequence and detaches

RNA polymerase

Newly made RNA

Direction of transcription Template

strand of DNA

RNA nucleotides

Terminator DNA

DNA of gene

RNA polymerase

Initiation

Promoter DNA

1

Elongation 2

Area shown in Figure 10.9A

Termination 3 Growing

RNA

RNA polymerase

Completed RNA

RNA PROCESSING• Messenger RNA (mRNA) - contains

codons for protein sequences

• Introns - interrupting segments

• Exons - coding segments

• Processing

• Cap added to the 5’ end (guanine nucleotides)

• Tail added to the 3’ end (Poly-A tail - 50-250 adenines)

• RNA Splicing - removal of introns and joining of exons

RNA transcript with cap and tail

Exons spliced together

Introns removed

Transcription Addition of cap and tail

Tail

DNA

mRNA

Cap

Exon Exon Exon Intron Intron

Coding sequence Nucleus

Cytoplasm

10

11

12

TRANSLATION

• Transfer RNA (tRNA) - match amino acids with the mRNA codon

• Each tRNA carries a specific amino acid

• Anticodon allows tRNA to bind to the complimentary codon on mRNA (A pairs with U; G pairs with C)

Anticodon

Amino acid attachment site

RNA polynucleotide chain

Hydrogen bond

TRANSLATION• Occurs at the ribosome (rRNA) in the

cytoplasm

• Three stages:

• Initiation: mRNA binds to ribosome and first tRNA at the start codon (P site)

• Elongation: next tRNA binds to mRNA at the A site and the two amino acids are joined as the ribosomes moves the molecules over to the next site

• Termination: stop codon is reached, all of the components are released

Polypeptide

A site

1 Codon recognition Codons

Amino acid

Anticodon P site

mRNA

2 Peptide bond formation

3 Translocation

New peptide bond

Stop codon

mRNA movement

Small ribosomal subunit

Start codon

P site

mRNA

A site

Large ribosomal subunit

Initiator tRNA

Met Met

2 1

OVERVIEW OF TRANSCRIPTION AND TRANSLATION

mRNA is transcribed from a DNA template.

RNA polymerase

Each amino acid attaches to its proper tRNA with the help of a specific enzyme and ATP.

Amino acid

DNA Transcription

mRNA

tRNA ATP

Translation

Enzyme

The mRNA, the first tRNA, and the ribosomal sub-units come together.

Initiator tRNA

Large ribosomal subunit

Anticodon

Initiation of polypeptide synthesis

Small ribosomal subunit

mRNA

Start Codon

1

2

3

New peptide bond forming Growing

polypeptide

4

A succession of tRNAs add their amino acids to the polypeptide chain as the mRNA is moved through the ribosome, one codon at a time.

Elongation

Codons

mRNA

Polypeptide

5 The ribosome recognizes a stop codon. The polypeptide is terminated and released.

Termination

Stop codon

13

14

15

MUTATIONS• Mutation - change in nucleotide

sequence

• Base substitutions: replacement of one nucleotide with another

• Could have no effect or would effect only one amino acid

• Deletions or insertions

• Can alter the reading frame and thus have significant impacts downstream

• Can be spontaneous (due to errors in replication or recombination) or induced by mutagens (radiation or chemicals)

Normal hemoglobin DNA Mutant hemoglobin DNA

Sickle-cell hemoglobin Normal hemoglobin

mRNA mRNA

Val Glu

Substitutions

Normal gene

Protein

Base substitution

Base deletion Missing

mRNA

Met Lys Phe Ser Ala

Met Lys Phe Gly Ala

Met Lys Leu Ala His

Insertions or Deletions

MICROBIAL GENETICS: VIRUSES• Two types of

reproductive cycles:

• Lytic: viral particles are produced using host cell components and the host cell dies when the viruses are released

• Lysogenic: viral DNA is inserted into the host by recombination

Bacterial chromosome

Phage injects DNA

Phage

Phage DNA

Attaches to cell

2

1

3

Phage DNA circularizes

Lytic cycle

4

New phage DNA and proteins are synthesized

Phages assemble

Cell lyses, releasing phages

6 5

7

Phage DNA inserts into the bacterial chromosome by recombination

Lysogenic bacterium reproduces normally, replicating the prophage at each cell division

Prophage

Lysogenic cycle

Many cell divisions

OR

ENVELOPED VIRUSES

Plasma membrane of host cell

VIRUS

Entry

Uncoating

Viral RNA (genome)

Viral RNA (genome)

2

1

3

Membranous envelope

Protein coat Glycoprotein spike

RNA synthesis by viral enzyme

Template

RNA synthesis (other strand)

Protein synthesis

mRNA

4 5

6

New viral genome

New viral proteins

Assembly

7

Exit

16

17

18

RETROVIRUSES

• HIV (human immunodeficiency virus)

• Contains two copies of its RNA genome and an enzyme, reverse transcriptase, which can produce DNA from an RNA template

Reverse transcriptase

RNA (two identical strands)

Protein coat

Glycoprotein Envelope

Double- stranded DNA

Viral RNA and proteins

DNA strand

Viral RNA

NUCLEUS

CYTOPLASM

Chromosomal DNA

Provirus DNA

RNA

2

1

5

3

4

6

VIROIDS AND PRIONS

• Viroid - circular RNA that infects plants

• Prions - infectious proteins that cause nervous system disorders in animals

• Misfolded forms of normal proteins that can convert other proteins

BACTERIA• Three ways they transfer

DNA

• Transformation - uptake of DNA from the environment

• Transduction - gene transfer through bacteriophages

• Conjugation - transfer of DNA from a donor to a recipient through a cytoplasmic bridge

Phage

Fragment of DNA from another bacterial cell (former phage host)

DNA enters cell

Bacterial chromosome (DNA)

Fragment of DNA from another bacterial cell

Mating bridge

Sex pili

Donor cell (“male”)

Recipient cell (“female”)

Transformation

Transduction

Conjugation

19

20

21

PLASMIDS

• Small circular DNA molecules that are separate from the bacterial chromosome

Male (donor) cell

Bacterial chromosome

F factor starts replication and transfer

F factor (plasmid)

Plasmid completes transfer and circularizes

Cell now male

22