1. Genes and How They Work
Chapter 15

2. The Nature of Genes
Early ideas to explain how genes work came from studying human diseases
Archibald Garrod – 1902
Recognized that alkaptonuria is inherited via a recessive allele
Proposed that patients with the disease lacked a particular enzyme
These ideas connected genes to enzymes

3. Beadle and Tatum – 1941
Deliberately set out to create mutations in chromosomes and verify that they behaved in a Mendelian fashion in crosses
Studied Neurospora crassa
Used X-rays to damage DNA
Looked for nutritional mutations
Had to have minimal media supplemented to grow

4. Beadle and Tatum looked for fungal cells lacking specific enzymes
The enzymes were required for the biochemical pathway producing the amino acid arginine
They identified mutants deficient in each enzyme of the pathway
One-gene/one-enzyme hypothesis has been modified to one-gene/one-polypeptide hypothesis

6. Central Dogma First described by Francis Crick
Information only flows from
DNA → RNA → protein
Transcription = DNA → RNA
Translation = RNA → protein
Retroviruses violate this order using reverse transcriptase to convert their RNA genome into DNA

7. RNA All synthesized from DNA template by transcription
Messenger RNA (mRNA)
Ribosomal RNA (rRNA)
Transfer RNA (tRNA)
Small nuclear RNA (snRNA)
Signal recognition particle RNA
Micro-RNA (miRNA) 7

8. Genetic Code DNA encoded amino acid order
Codon – block of 3 DNA nucleotides corresponding to an amino acid
Introduced single nulcleotide insertions or deletions and looked for mutations ---Frameshift mutations
Indicates importance of reading frame

9. Marshall Nirenberg identified the codons that specify each amino acid
Code is degenerate, meaning that some amino acids are specified
by more than one codon

10. Code practically universal
Strongest evidence that all living things share common ancestry
Advances in genetic engineering
Mitochondria and chloroplasts have some differences in “stop” signals

11. Prokaryotic transcription
Single RNA polymerase
Initiation of mRNA synthesis does not require a primer
Requires
Promoter
Start site Transcription unit
Termination site

12. Promoter Forms a recognition and binding site for the RNA polymerase
Found upstream of the start site
Not transcribed
Asymmetrical – indicate site of initiation and direction of transcription

13. Prokaryotic RNA polymerase Upstream
Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
TATAAT– Promoter (–10 sequence)  
Holoenzyme
Core
enzyme
Downstream  5׳ ’ 3׳ 
Start site (+1)
Template strand
TTGACA–Promoter
(–35 sequence)
Coding strand
Prokaryotic RNA polymerase
Upstream 5׳ 3׳ a. b. 13

14. Recognize specific signal in DNA
Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
 binds to DNA 
TATAAT– Promoter (– 10 sequence) 
Core
enzyme
Holoenzyme 
Downstream 5׳ 5׳ 9 3׳ 3׳ 
Core enzyme
Start site (+1) σ
TTGACA – Promoter
(–35 sequence)
Helix opens at
– 1 0 se q uence
Template
strand
Coding
strand
Prokaryotic RNA polymerase
Upstream 5׳ 3׳ 5׳ 3׳ a. b. 14

15. Initiation 15  TATAAT– Promoter (–10 sequence)  Holoenzyme Core
Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. 
TATAAT– Promoter (–10 sequence) 
Holoenzyme
Core
enzyme 
Downstream 5׳ 9 3׳ 
Start site (+1)
Template
strand
TTGACA–Promoter
(–35 sequence)
Coding
strand
Prokaryotic RNA polymerase
Upstream 5׳ 3׳ a. b.
Initiation
 binds to DNA
RNA polymerase bound
to unwound DNA
Transcription
bubble 5׳ 3׳
 dissociates
ATP
Helix opens at
–10 sequence
Start site RNA
synthesis begins 5׳ 3׳ 15

16. Elongation
Transcription bubble – contains RNA polymerase, DNA template, and growing RNA transcript

17. Termination Terminator sequences: G-C base-pairs --- A-T base-pairs-
 phosphodiester bonds (RNA-DNA) in the GC regions called hairpin, (RNAP stop)
 4 or ＞4 U (A-U is the weakest of the 4 hybrid base pairs, cannot hold the hybrid strands)
 RNA dissociates from the DNA
Signal hairpin AU release

18. Prokaryotic transcription is coupled to translation
Operon
1. Grouping of functionally related genes
2. Multiple enzymes for a pathway can be regulated together

19. Eukaryotic Transcription
3 different RNA polymerases
RNA polymerase I transcribes rRNA
RNA polymerase II transcribes mRNA and some snRNA
RNA polymerase III transcribes tRNA and some other small RNAs
Each RNA polymerase recognizes its own promoter

20. Initiation of transcription
Requires a series of transcription factors
Necessary to get the RNA polymerase II enzyme to a promoter and to initiate gene expression
Interact with RNA polymerase to form initiation complex at promoter
Termination
Termination sites not as well defined

21. Eukaryotic Transcription
3 different RNA polymerases
RNA polymerase I : rRNA
2.RNA polymerase II: mRNA and some snRNA
3.RNA polymerase III : tRNA and some other small RNAs
Each RNA polymerase recognizes its own promoter
Other transcription factors
Eukaryotic
DNA
Transcription
factor
TATA box

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23. 23 Other transcription factors RNA polymerase II Eukaryotic DNA
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Other transcription factors
RNA polymerase II
Eukaryotic
DNA
Transcription
factor
Initiation
complex
TATA box 23

24. mRNA modifications
In eukaryotes, the primary transcript must be modified to become mature mRNA
Addition of a 5′ cap
Protects from degradation; involved in translation initiation
Addition of a 3′ poly-A tail
Created by poly-A polymerase; protection from degradation
Removal of non-coding sequences (introns)
Pre-mRNA splicing done by spliceosome

25. 5´ cap HO OH P P P CH2 + 3´ poly-A tail 3´ N+ A A A A A A A CH3
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5´ cap HO OH P P P
CH2 +
3´ poly-A tail 3´ N+
A A A A A A A
CH3
Methyl group
A A U A A A
mRNA P P P G 5´
CH3

26. Eukaryotic pre-mRNA splicing
Introns – non-coding sequences
Exons – sequences that will be translated
Small ribonucleoprotein particles (snRNPs) recognize the intron–exon boundaries
snRNPs cluster with other proteins to form spliceosome
Responsible for removing introns

27. b: Courtesy of Dr. Bert O’Malley, Baylor College of Medicine
Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. E1 I1 E2 I2 E3 I3 E4 I4
DNA template
Exons
Transcription
Introns
5׳ cap
3׳ poly-A tail
Primary RNA transcript
Introns are removed
5׳ cap
3׳ poly-A tail a.
Mature mRNA
Intron 1
mRNA 3 2 4
DNA 7 5 6
Exon b. c. 27
b: Courtesy of Dr. Bert O’Malley, Baylor College of Medicine

28. 2. snRNPs associate with other factors to form spliceosome.
Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
snRNA
snRNPs
Exon 1
Intron
Exon 2 A 5´ 3´
Branch point A
1. snRNA forms base-pairs with 5´ end of intron, and at branch site.
Spliceosome A 5´ 3´
2. snRNPs associate with other factors to form spliceosome.
Lariat A 5´ 3´
3. 5´ end of intron is removed and forms bond at branch site,
forming a lariat. The 3´ end of the intron is then cut.
Excised
intron
Exon 1
Exon 2 5´
Mature mRNA 3´ 28
4. Exons are joined; spliceosome disassembles.

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30. Alternative splicing
Single primary transcript can be spliced into different mRNAs by the inclusion of different sets of exons
15% of known human genetic disorders are due to altered splicing
35 to 59% of human genes exhibit some form of alternative splicing
Explains how 25,000 genes of the human genome can encode the more than 80,000 different mRNAs

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