The Blueprint of Life, From DNA to Protein

The Blueprint of Life,From DNA to Protein

Chapter 7

Preview

• How does the genetic information pass on to the next generation?

• How is the information stored in DNA being used to make protein?

• How are the protein expression regulated?

The Blueprint of Life

• Characteristics of each cell dictated by information contained on DNA– DNA holds master blueprint

• All cell structures and processes directed by DNA

Review of DNA basics

5’ end (phosphate)3’ end (hydroxyl)

Two H bondsThree H bonds

•Double-stranded

•Double helix•Sugar-phosphate backbone

•Strands are complementary•Base-pairing rules:

A-TG-C

•Strands are anti-parallel

•Composed of deoxyribonucleotidesCovalently bonded in chains

N N N N N N N N N N5’ 3’

N N N N N N N N N N5’3’

If there are 400 cytosines in a DNA molecule that has 1000 base-pairs, how many adenines does the molecule have?

C C C C

G G G G

A A A A A A

T T T T T T

question

Figure 7.1

DNA Replication

DNA Replication

•Semi-conservative

Orig.

New

New

Orig.

DNA Replication

•Synthesis is 5’ 3’ (note: polymerase reads template 3’ 5’)

•Semi-conservative•Bi-directional

•DNA polymerase “reads” template, adds proper nucleotide to the 3’ end of the new chain

•Second round of replication can start before first is complete

•DNA polymerases generally corrects errors during replication (“proofreading”)Error rate = 1/billion nucleotides

•DNA polymerases require a primer (they can only add nucleotides onto an existing chain)

question

If a primer were available that bound to the center of the template molecule in the diagram below, which way would DNA polymerase move during DNA synthesis?

A G T C T G C C T A T C G T G A C T A5’ 3’

T C A G A C G G A T A G C A C T G A T5’3’ 5’

5’ 3’

5’

question



question

5’ 3’

5’



and so on…

question

5’ 3’

5’

DNA ReplicationReplication is initiated at a single distinct region (origin of replication = ori)

*Depicts only a small segment of the circular chromosome

5’

3’

3’

5’

DNA Replication

5’

3’

3’

5’

5’5’

Replication is initiated at a single distinct region (origin of replication = ori)

A short stretch of RNA (complementary to DNA) is synthesized

DNA Replication

5’

3’

3’

5’

5’

5’


DNA Replication

5’

3’

3’

5’

5’

5’

The replication fork (details are shown in Figure 7.6, which is optional)Leading strand - continuous synthesisLagging strand - discontinuous synthesis (Okazaki fragments)

DNA ligase


DNA Replication

•Semi-conservative•Bi-directional•Second round of replication can start before first is complete

DNA Replication

Gene expression

DNA to Proteins - General Principles

-- .. -.-. .-. --- -... .. --- .-.. --- --. -.--M I C R O B I O L O G Y

ATGCCCGTAGATGGCCCTGAGCGACCGGACCCTGATGCC

met pro val asp gly pro glu arg pro asp pro asp ala

Morse code: Distinct series of dots and dashes encode the 26 letters of the alphabet

Letters strung together make words sentences stories

DNA: Distinct series (triplets) of the four nucleotides encode the 20 amino acidsAmino acids strung together make proteins (structural and functional) cells organisms

Gene Expression - Overview

Coded by DNA:

Protein AProtein BProtein CProtein DProtein EProtein FProtein GProtein HProtein I

RNA transcripts:

Protein DProtein D

Protein DProtein D

Protein D

Protein D

Protein D

Protein D

Protein D

Protein D

Protein molecules

D

D

D

D

D

D

D

D

D

D

D

D

D

D

D

D

D

D

D

D

D

D

D

D

D

D

D

D

D

D

D

D

D

Transcription Translation

Gene: functional unit of DNA that contains information to produce a specific product

Gene Expression - Overview

Coded by DNA:


RNA transcripts:

Transcription

Messenger (mRNA)Ribosomal RNA (rRNA)Transfer RNA (tRNA)

rRNAtRNA

Three functional types of RNA:

Translation

Protein molecules

Review of RNA basics

•Composed of ribonucleotides (ribose not deoxyribose); uracil replaces thymine

Characteristics of RNA

OH




•Single-stranded•Sequence is “identical” to a stretch of one strand of DNA; complementary to the other




RNA




Template strand

RNA

Note: always read (and write) a DNA (or RNA) sequence in the 5’ to 3’ direction, or specify otherwise

Bacterial Gene Expression - Transcription

Transcription initiates at a promoter (sequence “theme” recognized by RNA polymerase)

Transcription stops at a terminator

5’TTGACA3’

3’AACTGT5’


Terms to note:

MonocistronicPolycistronic (prokaryotes only)

UpstreamDownstream

Initiation - RNA polymerase binds to promoter (guided by sigma factor)

Elongation - RNA polymerase synthesizes RNA in 5’ 3’ (no primer needed)

Termination -


5’ A T G A T C T G A G T A T G C G C T 3’

3’ T A C T A G A C T C A T A C G C G A 5’



5’ A T G A T C T G A G T A T G C G C T 3’

3’ U A C U A G A C U C A U A C G C G U 5’

5’ A U G A U C U G A G U A U G C G C U 3’


5’TTGACA3’

3’ -----------5’

5’-----------3’

3’ACAGTT5’

Prokaryotic Gene Expression - Transcription

Bacterial Gene Expression - Translation

•Ribosomes “read” mRNA; facilitate conversion of the encoded information into proteins•Message is read in triplets (codons)

AGAAUGCCCAAUGCGUUACGAUGCCC


•Ribosomes “read” mRNA; facilitate conversion of the encoded information into proteins•Message is read in triplets (codons)

But where should the ribosome start “reading”???

•Genetic code is degenerate



But where should the ribosome start “reading”???

•Prokaryotes (monocistronic and polycistronic messages) - translation begins at first AUG after a ribosome-binding site

•Ribosomes “read” mRNA; facilitate conversion of the encoded information into proteins

•Eukaryotes (moncistronic messages only) - translation begins at first AUG

•Message is read in triplets (codons)•Genetic code is degenerate



Proper reading frame is critical


AUG


Proper reading frame is critical



tRNAs are the “keys” that decipher the code

•Each tRNA carries a specific amino acid•Each tRNA has a specific anticodon, complementary to a codon, that binds mRNA


translocation

elongation factors

Initiation

Elongation

5’

E P A


Termination

Eukaryotic Gene Expression

Prokaryotic Gene Expression


AAAAAAAAAA

AAAAAAAAAA

Regulation of Gene Expression

• Microorganisms regulate its gene expression to adapt environment change– Controls metabolic pathways

• Two general mechanism– Allosteric inhibition of enzymes– Controlling synthesis of enzymes

» Directed at making only what is required

Prokaryotic Gene Regulation

Coded by DNA:


RNA transcripts:

Protein DProtein D

Protein DProtein D

Protein D

Protein D

Protein D

Protein D

Protein D

Protein D

Protein molecules

D

D

D

D

D

D

D

D

D

D

D

D

D

D

D

D

D

D

D

D

D

D

D

D

D

D

D

D

D

D

D

D

D

Transcription Translation

rRNAtRNA


Constitutive enzymes

Inducible enzymes

Repressible enzymes

Always produced

Genes turned “on” only when needed

Genes turned “off” when not needed

• Mechanisms controlling transcription– Often controlled by regulatory region near

promoter• Protein binds to region and acts as “on/off” switch

– Binding protein can act as repressor or activator» Repressor blocks transcription» Activator facilitates transcription



• Repressors– inhibits gene expression and decreases the

synthesis of enzymes– usually in response to the overabundance of

an end product– Repressors block the ability of RNA polymerase to bind

and initiate protein synthesis– Corepressor– inducer


• Activators– turns on the transcription of a gene or set of

genes• Inducer• Enzymes synthesized in the presence of inducers

are called inducible enzymes


• Operon model of gene expression– a set of genes that are controlled by

regulatory proteins – divided into two regions, the control region

and the structural region• The control region include the operator and the

promoter– The operator acts as the “on-off” switch

• The structural region includes the structural genes– This region contains the genes being transcribed

Operon structure

Promoter – Binding site for RNA polymerase

Operator – binding site for the repressor protein for the regulation of gene expression

Structural Genes – DNA sequence for specific proteins

Operator

Gene 1 Gene 3Gene 2

Promoter


DNA-binding proteins

repressor binds, blocking transcription

activator binds, facilitating transcription

(negative control)

(positive control)

Activity of activators/repressors can be controlled

Lac operon -galactosidase

transport

Lactose glucose + galactose

Lac operon -galactosidase

transport

Turned “on” only when lactose is present AND glucose levels are low

Is lactose present? If no, repress

Is glucose present? If yes, don’t activate

Lactose glucose + galactose

Lac operonTurned “on” only when lactose is present AND glucose levels are low

Glucose transport into cell lowers cAMP levels

Negative control - repressor is active if lactose is absent; inactive if lactose is present

Positive control - CAP only binds if cAMP is available (glucose levels are low)

The Blueprint of Life, From DNA to Protein

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