Transcript Biol 178 Lecture 29

Bio 178 Lecture 29
DNA 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.)
DNA Pol III is the main polymerase.
• Some of the “Other” Enzymatic Activities
1. DNA Helicase
Opens the DNA in front of polymerase.
2. DNA Primase
Synthesizes primers (RNA).
3. DNA pol I
Removes the primers (*5 ´ to 3´ exonuclease) and replaces
them with DNA.
4. DNA Ligase
Ligates Okazaki fragments (phosphodiester bond).
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 Eukaryotes
Similar to E. coli, but involves multiple origins of
replication for each chromosome.
One Gene/One Polypeptide
One 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 Dogma
DNARNAProtein
(The Expression of Genes)
• Stages of Protein Synthesis
1. 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
• Ribosomes
Composition
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 RNA
1. Ribosomal RNA (rRNA)
Site of ribosome where polypeptide is assembled.
2. Messenger RNA (mRNA)
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 Code
How Does the Order of DNA Nucleotides Specify the
Order of Amino Acids in Polypeptide?
• Crick et al., 1961
Hypothesis
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.
Results
Nonsense message was obtained when 1 or 2 nucleotides
were deleted (reading frame shifted).
3 deletions - reading frame restored & downstream
sequences transcribed correctly.
Conclusions
This 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.
• Prokaryotes
Transcription
1. 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.