chapter 15 genes and how they work. the nature of genes early ideas to explain how genes work came...

Chapter 15

Genes and How They Work

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

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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

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• 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

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

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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)

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Genetic Code

DNA encoded amino acid orderCodon – block of 3 DNA nucleotides corresponding to an amino acidIntroduced single nulcleotide insertions or deletions and looked for mutations ---Frameshift mutations

Indicates importance of reading frame

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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

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

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Prokaryotic transcription

• Single RNA polymerase

• Initiation of mRNA synthesis does not require a primer

• Requires– Promoter – Start site Transcription unit– Termination site

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• 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

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Upstream

Downstream

׳5 ׳3

b.

Coding strand

Template strand

׳5׳3

Start site (+1)

TATAAT– Promoter (–10 sequence)

TTGACA–Promoter (–35 sequence)

Holoenzyme

’

a.

Prokaryotic RNA polymerase

Coreenzyme

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Upstream

Downstream

׳5 ׳3

binds to DNA

b.

Helix opens at– 1 0 se q uence

Codingstrand

Templatestrand

׳5 ׳3

׳5׳3

Start site (+1)

TATAAT– Promoter (– 10 sequence)

TTGACA – Promoter (–35 sequence)

׳5׳3

Holoenzyme

9

a.


Coreenzyme

Recognize specific signal in DNA

σCore enzyme

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Upstream

Downstream

׳5 ׳3

b.

Codingstrand

Templatestrand

׳5׳3

Start site (+1)

TATAAT– Promoter (–10 sequence)

TTGACA–Promoter (–35 sequence)

Holoenzyme

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a.


Coreenzyme

binds to DNA

dissociates

ATP

Start site RNAsynthesis begins

Transcriptionbubble

RNA polymerase boundto unwound DNA

Helix opens at–10 sequence

׳5 ׳3

׳5׳3


Initiation

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

Elongation

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Termination

1. Terminator sequences: G-C base-pairs --- A-T base-pairs-

22 phosphodiester bonds (RNA-DNA) in the GC regions called hairpin, (RNAP stop)

22 4 or ＞ 4 U (A-U is the weakest of the 4 hybrid base pairs, cannot hold the hybrid strands)

22 RNA dissociates from the DNA

Signal hairpin AU release

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Prokaryotic transcription is coupled to translation

Operon1. Grouping of functionally related genes

2. Multiple enzymes for a pathway can be regulated together

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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

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• 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

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TATA box

Transcriptionfactor

EukaryoticDNA

Other transcription factors

Eukaryotic Transcription3 different RNA polymerases

1. RNA polymerase I : rRNA2.RNA polymerase II: mRNA and

some snRNA3.RNA polymerase III : tRNA and

some other small RNAs

Each RNA polymerase recognizes its own promoter

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Other transcription factors RNA polymerase II

TATA box

Transcriptionfactor

EukaryoticDNA

Initiationcomplex

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• 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

mRNA modifications

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A A A A A A A

Methyl group

3´ poly-A tail

5´ cap

CH2

HO OH

P P P

G

mRNA

PP

P

+N+

CH35´

3´

CH3

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

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E1 I1 E2 I2 E3 I3 E4 I4

Transcription

Introns are removed

Intron

Exon

DNA

1

2 34

5 67

a.

b. c.

Exons

Introns

mRNA

Mature mRNA

cap ׳poly-A tail5 ׳3

cap ׳5 poly-A tail ׳3

Primary RNA transcript

DNA template

b: Courtesy of Dr. Bert O’Malley, Baylor College of Medicine

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snRNPsExon 2Exon 1 Intron

Branch point A

snRNA

Exon 1 Exon 2

Lariat

5´

5´

3´

3´

5´

5´

3´

3´

2. snRNPs associate with other factors to form spliceosome.

4. Exons are joined; spliceosome disassembles.

1. snRNA forms base-pairs with 5´ end of intron, and at branch site.

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.

Mature mRNA

Excisedintron

SpliceosomeA

A

A

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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chapter 15 genes and how they work. the nature of genes early ideas to explain how genes work came...

Documents

rna proteinretroviruses

rna polymerasefound

rna genome

dna rna translation

site of initiation

binding site

tataat promoter

dna nucleotides