Bio 178 Lecture 29DNA and Gene Expression

Reading

• Chapters 14 & 15

Quiz Material

• Questions on P 300 & 318

• Chapters 14 & 15 Quizzes on Text Website (www.mhhe.com/raven7)

Outline• DNA

DNA Replication

• Gene Expression

Central Dogma

Major Players

Genetic Code

Transcription

Leading and Lagging Strands

The Replication Process (Cntd.)

• Some of the “Other” Enzymatic Activities

2. DNA PrimaseSynthesizes primers (RNA).

4. DNA LigaseLigates Okazaki fragments (phosphodiester bond).

3. DNA pol IRemoves the primers (*5 ´ to 3´ exonuclease) and replaces them with DNA.

1. DNA HelicaseOpens the DNA in front of polymerase.

DNA Pol III is the main polymerase.

Why Can’t DNA Pol I Remove Primers as a 3 to 5 Exonuclease?

E. Coli DNA Replication Enzymes

The DNA Replication Fork

DNA Replication

McGraw-Hill Video

DNA Replication in EukaryotesSimilar to E. coli, but involves multiple origins of replication for each chromosome.

One Gene/One PolypeptideOne gene encodes one polypeptide.

Proteins are Composed of Distinct Amino Acid Sequences• Sanger, 1953

• Sequenced insulin & showed that proteins are composed of specific amino acid sequences that are always the same for that protein.• This, and other work, led to the realization that the gene (specific sequence of nucleotides) determines the amino acid sequence of a protein.

Gene Expression - The Central DogmaDNARNAProtein

(The Expression of Genes)

• Stages of Protein Synthesis1. Transcription

DNA mRNA

Occurs in the nucleus.

2. Translation

mRNA Polypeptide Chain

Occurs in the cytoplasm.

Gene Expression - The Central Dogma

Gene Expression - The Major Players• RibosomesComposition

1. 2 subunits - a small and a large:

Eukaryotes - 40S and 60S.

Prokaryotes - 30S and *50S.

2. Small subunit (Prokaryotes):

> 20 proteins and 1 rRNA strand.

3. Large Subunit:

> 30 proteins and 2 rRNA strands.

Structure of Large Ribosomal Subunit (Prokaryotes)

Gray = RNA Gold = Protein

Ribosomes (Cntd.)Ribosomes are Ribozymes

Active site is composed of RNA. The proteins act as a scaffold.

Important Sites

E, P, and A - where tRNAs bind.

Gene Expression - The Major Players (Cntd.)

• Types of RNA1. Ribosomal RNA (rRNA)

2. Messenger RNA (mRNA)

Site of ribosome where polypeptide is assembled.

The single stranded RNA copy of the DNA that carries information from the nucleus to the cytoplasm.

3. Transfer RNA (tRNA)

Adapter molecules that pick up specific amino acids and pair them with the complementary sequence of nucleotides on the mRNA.

Transfer RNA Structure

The Genetic CodeHow Does the Order of DNA Nucleotides Specify the

Order of Amino Acids in Polypeptide?

• Crick et al., 1961Hypothesis

Genetic code consists of blocks of information (codons).

Each codon probably consists of 3 nucleotides that code for 1 amino acid.

Reasoning

4 bases - If only 2 bases compose a codon, there are 42 (16) possibilities. If 3 bases compose a codon there are 43 (64) possibilities. There are 20 aa 3 bases/codon would be enough.

Genetic Code - Crick et al. (Cntd.)Question

Is the genetic code continuous?

Experiment

Deleted 1, 2, or 3 nucleotides at a time from viral DNA and tested the effect on transcription.ResultsNonsense message was obtained when 1 or 2 nucleotides were deleted (reading frame shifted).3 deletions - reading frame restored & downstream sequences transcribed correctly.ConclusionsThis is a triplet code with no “puctuation”.

The Genetic Code (Cntd.)

• Breaking the Code

Nirenberg (1961)

PolyU mRNA translated in vitro Polyphenylalanine

UUU codes for phenylalanine.

Nirenberg and Leder (1964)

Developed an assay (involving radioactive amino acids) identified what aa 47 of the 64 triplets code for.

H. Gobind Khorana

Decoded the remaining 17 triplets.

The Genetic Code

The Genetic Code (Cntd.)• Universality

Almost all organisms use the same genetic code.

Strong evidence for a shared common evolutionary lineage.

Important for genetic engineering.

• Exceptions

Mitochondria & chloroplasts - Suggests the change occurred after they became endosymbiotic.

Certain protists.

Transcription• Prokaryotes1. RNA Polymerase

A large enzyme that:

(a) Binds to the promoter.

(b) Moves down the DNA, untwisting it and breaking the hydrogen bonds between the bases.

(c) Pairs complementary nucleotides to the template strand (antisense (-)). Synthesis occurs 5 to 3 .

(d) Detaches at the termination site.

Transcription Bubble

Transcription in Prokaryotes (Cntd.)

2. Promoter

A short sequence where RNA pol binds eg. TTGACA.

Is not transcribed.

Variable efficiency.

3. Elongation

No primer.

First nucleotide usually ATP or GTP.

Transcription bubble moves ~ 50 nts/sec.

Transcription Initiation

Transcription (Cntd.)

4. Termination

At the end of the gene “stop” sequences cause transcription to stop, the RNA-DNA hybrid to dissociate, & the DNA in the transcription bubble to rewind.

Example of a Stop Signal:

Series of GC base pairs, followed by series of AT base pairs GC hairpin followed by a polyU sequence.

Hairpin causes RNA pol to pause & the pairing of U with A is weak Dissociation.