Chapter 8

Genetics and Molecular Biology

Introduction

DNA contains info on what makes a cell a cell

“Blueprint” of the cell; nucleic acid• molecule that carries info that determines the

traits/characteristics of the cell/organism• This info determines the characteristics (traits) of cells–

shape, structural features, metabolism, growth characteristics, ability to cause disease, etc

• During reproduction, Info in the DNA is copied and passed from one generation to the next: “genetic”– Also called “genetic material”

From DNA to traits

Genetic info is “written” as a code; cell has to decode this info in order to turn this info into traits

Genetic info = referred to as Genetic code

What does this coded info really mean???

From DNA to traits

Genetic info/code contained in DNA is basically information about:• all the proteins that are needed by the cell- enzymes, ribo prot, prot that make up cell components• ribosomal RNA• tRNA- RNA tool involved in protein synthesis

expression: turn genetic info in DNA into trait• Using the info in DNA to make these 3 types of “fctnal products”• once products are made, perform fctn in cell and results in observable characteristics

Genetic info in DNA used for making proteinsProteins serve as enzymes or cell structure/partsProteins contribute to cell’s characteristics via enzyme activity

and by being part of the cell’s structure• Much of cell’s structural components (eg, flagella, capsule)

are made of proteins; structural characteristics determined by whether DNA contains info about protein components

--eg, Bacteria that can produce exotoxin- contains info in its DNA about the exotoxin

• ENZYMES are also proteins; responsible for biochemical traits

-- ability to do certain reactions due to DNA containing info about specific enzymes

-- responsible for many traits not directly linked to proteins

• Observable traits of cells/organisms are determined by the genetic information they have (in their DNA)– Info allows them to make cell components or

enzymes that result in the trait

• To turn genetic info into trait, cell “converts” the info in DNA into instructions on how to make proteins. Ribosome follow these instructions to make proteins– proteins perform functions in the cell; results in a

“trait” that we can observe

Summary of CH 8“Genetics”: study of how DNA carries info, how it is

replicated/copied and how info is expressed as traits

• Structure of DNA

• Replication: how cell makes a copy DNA to pass genetic info to the next generation

• Expression: How cells read info in DNA to make functional products that result in traits

• How cells turn on and turn off “expression” of genetic information

• Changes in genetic info and their effects

Definitions!

• Gene – segment of DNA that encodes for a single functional product: protein, rRNA or tRNA

• Genome– all the genetic info contained in the cell; all the DNA molecules the cell has

• Genotype – the genetic makeup; specific genetic information that a cell/organism has

• Phenotype – the physical trait (determined by genotype)

• Expression– using genetic info to make a functional product [and produce observable trait]

Forms of DNA: chromosome

• Chromosome – long DNA molecules that codes for the cell’s vital, defining characteristics (things/proteins that cell absolutely needs)

• “vital” DNA plus proteins associated w/ this DNA • Bact has a single chromosome (circular and supercoiled)-

about 5 million nucleotides– Contains tens of thousands of genes (info about tens of

thousands of functional products)

• Faithfully copied and passed to next generation- copied ONLY when the cell divides

Forms of DNA: plasmids

• Plasmids – circular pieces of DNA that are separate and distinct from chromosome

• Replicate independently of chromosome; copied even when cell is not dividing

• Present only in microorgs (bact, yeast). • Carry genes that code for products not essential to

cell’s survival (eg antibiotic resistance, toxins, non essential structures: pili).

• Smaller/shorter than the chromosome; has fewer genes (dozen vs 10000+)

• Valuable “tool” in helping us to mass produce proteins (next chapter)

Forms of DNA: transposons

• Transposons – mobile segments of DNA; genes that can move from place to place on the DNA.

Review of DNA

• 2 polymers/chains of nuctides wrapped around each other

• Each chain is made of many nucleotides joined together (polymer)

• H-bonds form b/w bases of the 2 strands

• Strands are antiparallel

Figure 8.4

WHAT IS DNA—Review of structure

Nucleotide-3 components: phosphate gp linked to sugar (deoxyribose) linked to a “base” (C and N atoms arranged into a “ring”)

DNA is polymer of nucleotides that contain deoxy-riboseLink a bunch of nucleotides together to form a chain/strand (polymer)– DNA made up of 2 strands wrapped round ea other

RNA also a polymer of nucleotides. But RNA nucleotides contain the sugar ribose and is single stranded

DNA nucleotide are nuctides that have the sugar deoxy-ribose

Join the sugar (deoxyribose)of this nucleotide to…

…the phsosphate group of this nucleotide

individual DNA nucleotides are linked together into a polymer or a DNA “strand” by covalent bonds between their sugars and phsophate groups

polymer of nuctides also called a “strand”

DNA is made of 2 “strands”, or polymers of DNA nucleotides

=

Backbone

vs bases

DNA Structure

2 strands wrap around each other-held together by hydrogen bonds between bases

+ =

DNA StructureLink nucleotides into a chain/polymerNucleotides joined at the sugar of one nucleotide and the

phosphate group (PO4) of anotherThe linked sugars and phosphates of

the chain is “sugar-PO4 backbone”2 DNA strands/chains wrap around each other: double helix or “twisted ladder”-rails and steps (DNA is a double stranded nuc acid)2 DNA strands are held together by H-bonds: bases on one strand form H-bonds with bases on the other strand

TwistFig. 10.4

A rope-ladder model of a double helix

One strand

another strand

backbone

base

DNA: four bases Adenine, Thymine, Cytosine, Guanine

Each base can form a H-bond only with a specific base on the other strand (Base pairing rules)• Adenine on one strand H-bonds to Thymine on the other strand (and vice versa), but not to Cytosine or Guanine• Guanine on one strand H-bonds to Cytosine on the other strand (and vice versa), but not to Thymine or Adenine

A pairs (forms H-bonds) w/ T, not G and CG pairs (forms H-bonds) w/ C, not T and A

Sequence of bases in DNA is the genetic code!!!Base sequence of DNA defines the aa sequence (and structure) of proteins

If we know the base sequence of one DNA strand, we can use base pairing rules to figure out the base sequence of its matching strand

Base sequence on one strand tells us the base sequence of its matching strand.

“complementarity”: each DNA strand can pair only with a strand that has matching bases in the right sequence

A DNA strand can serve as a template for its matching strand

Key to understanding genetics

• Key to understanding genetics is to understand “complementarity”

• Look at the base sequence of a DNA (or RNA) strand and you can tell which bases match this sequence

• GATTCAT

Key to understanding genetics

• Key to understanding genetics is to understand “complementarity”

• Look at the base sequence of a DNA (or RNA) strand and you can tell which bases match this sequence

• GATTCAT

CTAAGTA• Pairing up nucleic acid (DNA, RNA) strands with

matching base sequences is the key to genetics• (a DNA and a RNA strand can also pair up if

they have matching base sequences)

Each DNA strand has “direction”/distinct ends end where phosphate gp is not attached

to another nucleotide is 5’ end (5’ end- “front” end) end where sugar is not attached to another nucleotide is 3’ endThe 2 strands of DNA are in opposite orientations: “anti-parallel”(upside down w/ respect to ea other)

Base sequence written left to right from 5’ to 3’end: write base seq of 1 strand

Base Pairs (bp)

• 2 bases (on opposite strands) that are forming H-bonds with each other

• Unit of length in DNA-eg, this segment is 4 base pairs

• Gives you an idea of how long a gene is…

gene w/1200bp vs 4000bp

Summary of DNA structure

• DNA is double stranded• DNA has base-pairing rules- • A DNA strand can serve as the template for its

matching strand (if we know the base seq of one strand, we

can determine base seq of its matching strand)• Nucleic acid strands with matching bases can

bind to each other!!!!• Sequence of bases in DNA is the genetic code/

information– defines the amino acid sequence of proteins

• 5’ and 3’ ends of DNA

Summary of DNA structure

• DNA is double stranded• Bases on 1 strand form Hbonds w/ bases on

other strand- holds 2 strands together• DNA has base-pairing rules- A&T; G&C• DNA strand w/ a specific base sequence can

pair up only with another DNA strand that has matching bases

• A DNA strand can serve as the template for its matching strand (if we know the base seq of one strand, we

can determine base seq of its matching strand)• Sequence of bases in DNA is the genetic code/

information– defines the amino acid sequence of proteins

Replication-occurs during fission (cell division)

Figure 8.3

1. One parental DNA molc (dbl strand) becomes 2 “daughter” DNA molcs ; copying DNA

2. Outline: separate the strands of parent DNA

• Each strand is template for building a new matching strand (base seq read to make matching strand)

3. In daughter DNA:• One strand is from parent DNA• One strand is newly made (“daughter

strand”)“SEMICONSERVATIVE”

Closeup of DNA polymerization

Figure 8.5

• Polymerization: putting nucleotides together to make a nucleic acid strand• occurs during replication of DNA• one strand serves as the template for making its matching strand• application of base pairing rules- base sequence of the template strand is read; nucleotides with matching bases are joined together to form the other strand

5’

3’

5’

3’ 5’

3’5’

3’

Closeup of DNA polymerization

Figure 8.5

• DNA POL reads base seq of template strand from 3’ to 5’ end - puts together nuctides with matching base to make new strand• new DNA strand is made from 5’ to 3’ end - DNA POL can only add new nuctide to 3’ end of new strand - adds one nucleotide at a time• nucleoside triphosphate- raw material; nuctide w/ 2 extra phosphates!Requires E, provided by breaking bond b/w 1st and 2nd Phosphate

5’

3’

5’

3’ 5’

3’5’

3’

• Replication starts at “origin”--site where DNA strands are initially sep’d. sep of DNA strands spreads from origin

• A replication bubble will form as strand sep spreads• replication fork: edge of replication bubble

Replication Bubbles and Forks: where repl take place

Figure 8.7

Replication Mechanism

Figure 8.6

• DNA Helicase – unwinds DNA and separates 2 strnds• Single-stranded-DNA-binding protein (ssDNAbp)• Primase- makes short RNA chain (primer)• DNA Polymerase

– Synthesizes DNA: adds DNA nucleotides to primer to form new strand

– Works only 5 3 (adds nuctide to 3’end of new strnd)– Leading strand synthesized continuously, lagging strand

synthesized discontinuously • DNA ligase – glues together lagging strand fragments• Nucleoside triphosphate (nuctide w/ 2 addt’l PO4)=not

protein; raw materials that make up the new strand• All located in cytoplasm

Materials Needed in Replication

Gene Expression

A Gene: a segment of DNA that codes for a single polypeptide, tRNA or rRNA (contains information on how to make a fctnal product)

- 90% of genes in bacteria code for polypeptide

Gene Expression: using information encoded in the gene to make a fctnal product

Examine how gene is expressed to make a proteinOne gene codes for one polypeptide

Gene Expression to Make Protein

Turn info encoded in a gene into a polypeptide

(gene)

Transcription/trx: base seq of the gene is read to make mRNA w/ a complementary base sequence; gene DNA used as template to make mRNA

Translation/tln: mRNA is read by the ribosome to make protein- base seq of mRNA tells ribo which aa acids to join together.

CENTRAL DOGMA

2 stage process involved in xpr of genes that code for proteins• Cell uses/reads info in DNA to make mRNA:TRANSCRIPTION (rewrite the genetic info in DNA into instructions/mRNA for the cell)• mRNA then directs the cell to make the protein: TRANSLATION (cell reads the instructions/ mRNA to make a protein)

These instructions ultimately come from DNAGene expression: take info in DNA and “turn” it into protein

• DNA is transcribed to make RNA- in “central dogma”, to use the base sequence of DNA to make mRNA

• mRNA: made from DNA template

-- messenger RNA: instructions on how to make a polypeptide; derived from DNA(gene) base sequence

-- Base sequence matches that of DNA template– Single stranded polymer of RNA nuctides (sugar part is

ribose): phosphate-ribose(sugar)-base– 4 Bases: A,G,C (same as DNA). But Uracil instead of T– Synth’d by joining RNA nuctides w/ bases that match or

“pair up” with bases in the DNA template• 3 Steps: Initiation, Elongation, Termination

Transcription-to make RNA

Transcription

• ONE strand of the DNA serves as “template” for making mRNA

• cell reads DNA base sequence on one strand and puts together RNA nuctides with matching bases to make mRNA

How does cell know which RNA nuctides/bases to put into the mRNA???

Based on base pairing rules:

DNA template says: put in RNA nuctide w/ base: A U (equivalent of T in RNA) T A G C C G

Example: DNA sequence is: TAGACTMake mRNA w/ seq: AUCUGA i.e., base seq of mRNA is complementary to that of DNA template

Base sequence of mRNA specifies aa sequence of the polypeptide that’ll be made during TRANSLATION

Transcription: materials

RNAP: RNA polymerase- enzyme that makes mRNA by polymerizing (putting together) RNA nucleotides

RNA nucleotides: in the form of RNA nucleoside triphosphate (nucleotide w/2 addt’l phosphates)

breaking bonds b/w 1st 2 PO4 generates E needed for adding new RNA nuctide to mRNA

• Monomers that make up the mRNA; raw materials

New RNA nucleotides are added to 3’end of mRNA (made in 5’ to 3’ direction)

Prok/bacterial Gene has 2 major “regions”Coding region: portion of the gene that contains information

about the polypepetide; contains the bases that actually codes for the polypepetide; serves as template for making mRNA

Promoter: region in front of the coding region; RNAP has to bind to the promoter in order to initiate transcription; does not contain info that code for polypeptide

In transcription, the bases in the coding region serve as the template for making mRNA (promo not used as template to make mRNA); mRNA corresponds ONLY to reading frame

GENEpromoter Coding region/reading frame

Steps in Transcription

• Initiation – RNAP binds to promoter. DNA strands in front portion of coding region separate

• Elongation – RNAP moves into coding region; reads base sequence of the coding region makes mRNA

• Termination – RNAP reaches terminator and stops transcription; mRNA synthesis is completed

Steps in Transcription

What Happens After mRNA is made (after transcription)???

• In prokaryotes, once Trx is complete, mRNA undergoes translation right away (mRNA is read by ribosome)

• In eukaryotes, mRNA does NOT undergo translation right away– it has to be modified/ “processed” before it can undergo translation

-- cap and tail

-- splicing/editing

RNA processing/editing in Eukaryotes

• Prok: all bases in coding region of gene codes for protein

• Euk genes: bases that code for protein are interrupted by bases that don’t code for prot.

• Transcribe entire seq

• Then remove noncoding seq (intron)

• Splicing – introns are removed and exons spliced together

• Spliced product is exported from nucleus

Convention

DNA strand that serves as a template for making RNA is called the “ANTI-SENSE” strand

The complementary DNA strand is the “SENSE” strand

By convention, when writing a DNA sequence, you write the base sequence of the SENSE strand only, with the 5’ end on the left and 3’ end on the right

ATGCGAA Base sequence of sense strand is “equivalent” to base sequence of mRNA (except RNA uses U instead of T), since both sense strand and mRNA are complementary to anti-sense strand.

Translation and mRNA

• mRNA carries the instructions on how to make a protein (based on base sequence of DNA/gene)

• during TRANSLATION, ribosome reads base sequence of mRNA to make protein

• mRNA serves as “instructions” read by ribosome to make protein specified/encoded by DNA/gene

• mRNA is transient: not kept in the cell permanently; degraded after the protein has been made.

• Needs:– mRNA: instructions for

making protein– Ribosome– tRNA– Amino acids

• Steps– Initiation:binding to mRNA

– Elongation:joining aa’s

– Termination

Translation

Figure 8.2

Translation Needs mRNA

• Ribosomes read the base sequence of mRNA 3 bases at a time to make a protein

• Every 3 bases in mRNA codes for a particular amino acid

• codon: collection of 3 bases in mRNA that specifies an amino acid

• AUG GAU GCC GUC ACU codons are read sequentially from 5’ end to 3’ end of mRNA

• mRNA seq is read like a sentence w/ bunch of 3 lettered words in succession. Overall message of mRNA: amino acid sequence of the protein/polypeptide

• Base sequence of mRNA determines the aa seq of the protein: info ultimately comes from DNA/gene

The Genetic Code

• Meaning of the codons• Each triplet of bases on

mRNA codes for one amino acid (codon)

• Codon is like a 3 lettered word (each base is a letter)

• Meaning of word: aa• Degenerate: one amino

acid can be specified by several codons

Examples: GUA=??? ACU=???mRNA seq=aa seq

tRNA: transfer RNA

RNA molecule that brings the correct aa (the one defined by the codon in mRNA) to the ribosome

tRNA is a single strand RNA molecule (polymer of RNA nucleotides) that folds on itself into a shape like the letter “t”

One end has a sequence of 3 bases that matches a codon on mRNA.—”anti-codon”; can pair up w/ complementary codon

--allows tRNA to bind mRNA. tRNA anticodon forms H-bonds/pairs up with matching codon on mRNA.

Other end of tRNA is attached to the amino acid specified by the codon that matches tRNA’s anticodon

• An example: There is a tRNA that has the anticodon 3’UAC5’ on one endWhich pairs up with codon AUG on mRNA…the other end of this tRNA is attached to the aa

specified by codon AUG—methionine Base pairing/matching rules are the same in RNA

Complementarity b/w codon and anticodon is key to making a protein w/ right aa seq

(binding of tRNA to mRNA via interaction between the anticodon&codon allows the correct aa to be placed in the polypeptide)

• An example:

mRNA codon AUG

There is a tRNA with the matching anticodon

UAC on one end

…and on the other end, it’s attached to the aa specified by codon AUG—which is??

(quizzete)

Ribosomes

• Enzyme: catalyzes formation of peptide bond b/w aa’s

--takes aa’s and join them together to form polypep/Prot• Large (50S) and small (30S) subunits• Each subunit made of many protein and RNA molecules

--rRNA: “skeleton”; protein: “meat”• Antibiotics directed against bacterial ribosomes

Translation - Termination

Translation - Termination

Figure 8.10.8

Coupled TRx-Tln in Prokaryotes

Figure 8.11

Transcription and translation can occur simultaneously in bacteria (prok)i.e., mRNA undergoes tln WHILE it’s BEING made- tln can begin BEFORE trx is completed

Tln can occurwhile Trx is

IN PROGRESS !!!

Expression of genes that code for protein vs genes that code for rRNA or tRNA

• To express gene that codes for protein: gene undergoes transcription to make mRNA, which is then read by ribo to make protein (translation/tln)

• To express gene that codes for rRNA or tRNA: gene undergoes transcription ONLY- cell reads base seq of DNA to make rRNA or tRNA

-- once tRNA or rRNA is made, they perform fctn

Information in nucleic acids

• Information carried by nucleic acids is found in the base sequence

• Gene (DNA)- has meaning– Code for proteins: base seq of mRNA and aa seq of

polypeptide– Code for tRNA and rRNA: base seq of these RNA’s

• mRNA – has meaning; codon specifies an specific amino acid; instructions for ribosomes

• rRNA- base sequences are recognized by ribosomal proteins; allows ribo proteins to fit over the rRNA

Regulation of Transcription (making mRNA)

• Activation– An activator turns on transcription

• Repression– A repressor turns off transcription– An inducer removes the repressor

Regulation of Bacterial Gene Expression

Some terms• Constituitive gene: expressed all the time; code

for proteins needed all the time– TCA and glysis enzymes; ETS; SOD/catalase; ribo

proteins

• Regulated gene: expression can be turned ON or OFF depending on cell’s needs– Cell needs protein: expression turned ON; cell doesn’t

need protein: expression turned OFF

• Focus on how regulated genes are turned on and off

• In prokaryotes, regulation takes place at the transcriptional level- – expression is turned ON by allowing transcription to

occur– Expression is turned OFF by inhibiting transcription

• If transcription occurs, then translation occurs and the protein is made

• Activation: turn ON gene expression/trx• Repression: turn OFF gene expression/ trx• Repressor- molecule that shuts off trx• Inducer- molecule that prevents repressor from

working (allows trx to be turned on)• Operon: group of genes whose expression is

turned ON and OFF together– Genes of operon code for proteins that work together

to perform a common function (eg, enzymes of pathway)

– 2 examples: trp operon and lac operon– Repressor is key to regulation (turn xpr on and off)

Anatomy of operon

• Structural genes: reading frames; info about protein

• Control region: controls whether trx is ON or OFF– Promotor: binding site for RNA POL- has to bind to

promotor for transcription to occur

– Operator: binding site for repressor- shuts off TRX

• Repressor coded for by constitutive gene; not part of operon

Operons

Figure 8.14.1

Repressor not bound to OPER; trx can occur

REPRESSOR

Operons

Figure 8.14.1

REPRESSOR

Repressor binds to OPER: trx of struct. genes is turned “OFF” (trx doesn’t occur)

Trp Operon

Figure 8.14.1

5 genes that code for enzymes that make trp, needed for growth

Makes trp when there is no trp available in environment

If trp is available- cell takes in trp; doesn’t need to make its own

REPRESSOR

E D C B A

Trp Operon- when trp is absent

Figure 8.14.1

Trp not available most of the time: genes need to be expressed- default position of operon= ON

Repressor can’t bind to operator when trp is not present in the cell; transcription occurs when RNAPOL binds to promotor

REPRESSOR

E D C B A

Trp Operon- when trp is present

Figure 8.14.1

If trp is present- cell does not need to make trp; expression is turned OFF

Cell takes in trp, trp binds to repressor; repressor binds to operator; blocks RNAP from reaching the structural genes- transcription will not occur

REPRESSOR-Trp

E D C B A

Trp Operon- when trp is present

Figure 8.14.1

If trp is present- cell does not need to make trp; expression is turned OFF

Cell takes in trp, trp binds to repressor; repressor binds to operator; blocks RNAP from reaching the structural genes- transcription will not occur

REPRESSOR-Trp

E D C B A

The Lac Operon

• Background– if glucose is not present, bacteria will use

lactose as a carbon source– Three proteins are necessary to metabolize

lactose– Proteins coded by the 3 genes of lac operon

(Z,Y,A)

The Lac Operon

Figure 8.14.1

The Lac Operon

Figure 8.14.1

REPRESSOR

Repressor binds to OPER: trx of struct. genes is turned “OFF” (trx doesn’t occur)

Repressor of lac operon is different from the one used in trp operon!

The Lac Operon

Figure 8.14.1

When Repressor does not bind to OPER--trx can be turned on

REPRESSOR

Trx/Expression turns on ONLY when lactose is present AND the glucose is absent

(both conditions must be fulfilled for trx to be turned ON)

Expression of Lac operon is turned OFF most of the time: default position

Turned off b/c lactose NOT present most of the time; no need to make proteins involved in lactose metabolism

Why is absence of glucose(glc) necessary?• glc is “preferred” nutrient/E source for bact• when glc is available, bact will use ONLY glc

– bact will NOT use other nutrients (lactose) as long as glc is present

– bact doesn’t need enz that allow it to use lactose when glc is available -- no need to turn on trx/xpr of lac operon

• bact will use lactose only when glc is absent

ie- cell needs enz that allow it to use lac ONLY when glc is absent; trx/xpr of lac operon occurs only in the absence of glc

Why is presence of Lactose necessary?• If there is no lactose present- cell doesn’t need

enz that allow it to use lactose– no need to turn on xpr/trx of lac operon when lactose is absent

• Therefore, the enz that allow the cell to use lactose are needed ONLY when lactose is available hence, trx/xpr of lac operon occurs when lactose is present

Taken together, the only time that the cell needs the enz that allow it to use lac as a nutrient is when both of the following conds are met--glucose is absent AND lactose is present

• This is the only time that the xpr/trx of lac operon is turned ON…

When lac is Absent, Trx/expression of Lac Operon is Turned “OFF”

Figure 8.14.2

This is what happens when Lactose is Absent…

Repressor binds to OPER;Trx does not occur

Physically blocks RNAPol bound to PROMO from reaching the structural genes; no TRX

How is Trx of Lac operon turned ON???

Trx/Expression turns on ONLY in the presence of lactose AND the absence of glucose (both conditions must be fulfilled for trx to be turned ON)

First, look at what happens when glucose is absent

– The lac operon senses glucose and lactose levels

Activation at the Lac Operon

High glucose=low cAMP

or NO glucose

Low glucose=high cAMPNo glucose=v. high cAMP

In absence of glucose:

cAMP-CAP facilitates binding of RNAP to PromoRNAP 80x more likely to bind to promo that has camp-cap

No glucose: increase likelihood of RNAP binding to promo; incr frequency of transcription

Induction at the Lac Operon

Figure 8.14.4

When Lactose is present

Lactose binds to Repr and removes it from OPER; allows Trx to occur (RNAPol can now reach and read genes to make mRNA

ie., presence of lactose gets repressor out of the way (off the oper) so trx can occur

Lactose binds repressor

Induction at the Lac Operon

Figure 8.14.4

When Lactose is present

Lactose binds to Repr and removes it from OPER; allows Trx to occur (RNAPol can now reach and read genes to make mRNA

ie., presence of lactose gets repressor out of the way (off the oper) so trx can occur

Summary of Regulation at the Lac Operon

• How is trx of operon turned ON? Needs both absence of glucose- helps RNAP to bind to promo of lac operon AND presence of lactose-gets repressor out of the way– When glucose is absent, cAMP levels are very high– excess cAMP forms complex w/ CAP and binds to the

promoter, helping RNAP to bind promoter– When Lactose is present, it binds repressor, removing

it from operator– allows RNAP to transcribe genes

• default position is “OFF”- lactose absent most of the time--whenever lactose is absent, the repressor (I) binds to operator region of DNA, preventing trx

Significance of controlling gene expression

• Gene expression is closely related to pathogenesis

• Bact gets into the body- conditions inside the body causes bacteria to turn on certain genes

• Bacteria can make structures that allow it to establish residency in body (eg- invasins, various enzymes) and to damage cells (eg-exotoxins)

• Control disease by understanding which genes are expressed inside the host and the conditions that cause these genes to be expressed?

• Mutations: changes in base sequence of DNA

• Mutations are passed to subsequent generations, because the changes in bases will be copied faithfully in DNA replication (preexisting/parental DNA base sequence determines base sequence of new/daughter DNA)

ACTGTACGC change C to T ATTGTACGC

• Mutations lead to production of mRNA with changed base sequence during gene expression– may result in making proteins with the wrong amino acids and get a nonfunctional protein

Wildtype: “normal”; DNA base sequence without mutations or changes; cell or organism w/o mutations

Mutant: DNA with altered base sequence/ mutation (has base sequence different from wildtype DNA); cell or organism w/ mutations

Also applies to proteins (aa sequence)

• Change in the genetic info/base sequence of DNA. Mutations may be neutral, beneficial, or harmful

• Ways to change the base sequence of DNA– Deletion – remove bases– Insertion – add bases– Point – substitute one base

Mutation

Types of mutations according to effect on protein structure and/or function • Missense – replacement of one amino acid

by another• Nonsense – produces protein that has too

few aa’s; changes the codon that codes for amino acid into a stop codon.

• Frameshift– causes mRNA to be “misread”; results in multiple amino acid substitutions

• Silent – no change in amino acid sequence of the protein

Silent Mutation

WT DNA Mut DNA

AAC GAC – A changed to G trx trx

UUG-in mRNA CUG- in mRNA

=leu also leu due to degeneracy

NO change in aa comp of the protein despite base substitution- no change in struct and fctn of protein

Missense Mutation

Figure 8.17a, b

Change this nuctide w/ C to nuctide w/T: codon becomes AGC instead of GGC specifies ser instead of gly

Base substitution results in the replacement of one aa by another: changes codon sequence in mRNA so that the mutant codon specifies a different aa

Mutation causes gly to be replaced by ser (changes the mRNA codon)

Missense Mutation

WT DNA Mutant DNA

CCG TCG- C changed to T

GGC- in mRNA AGC- in mRNA

=gly =ser

Mutation cause gly to be replaced by ser in the protein

Sickle cell anaemia: • single missense mutaion in gene coding for

haemoglobin (substitution of just one base: an A by a T) changes 6th amino acid

• results in replacement of a glutamic acid by valine in haemoglobin; changes shape of haemoglobin and shape of RBC—can’t carry oxygen thru small capillaries

• People w/ mutant (sickle cell) gene are more resistant to malaria

Nonsense Mutation

Figure 8.17a, c

Change “T” to “A”: changes mRNA codon from AAG (lys) to UAG (stop) Ribosome stops translation prematurely (transl stops after 1st amino acid)

Base substitution results in making polypeptide with too few aa• substitution changes codon seq from one that specifies an aa to a stop codon that halts translation

TTCATC

AAGUAG

lys--->stop

Deletion and Insertion can result in a Frameshift

Figure 8.17a, d

Remove/delete this nuctide w/ A: codon UUU becomes UUG; all subsequent codons also changed

Ribo reads UUG instead of UUU

Codons are shifted—changes the way ribosome reads the mRNA. Causes multiple amino acid substitutions

Thefatcatatethetinbat

Thefatcattethetinbatsentence w/ a removed

• mRNA is a sentence w/ 3-lettered words• Thefatcatatethetinbat= read 3 letters at a time• Thefatcatatethetinbat= remove the “a”• Thefatcattethetinbat= sentence w/ a removed; changes

all the words AFTER the deletion site

• Thefatcatatethetinbat- let’s add the letter “g”• Thefatcatgatethetinbat

• Adding or deleting bases changes the 3 lettered words in the sentence- changes the meaning of the sentence

• If this occurs in mRNA- it changes the base sequence of the codons in the mRNA and results in multiple aa substitutions

• DNA-- TAC TTC AAA CGC ATT• RNA– 5’AUG AAG UUU GCG UAA 3’ protein met lys phe ser stop

• Take away 3rd A in “AAA”• DNA TAC TTC AA-C GCA TT• mRNA 5’AUG AAG UU-G CGU AA 3’ protein met lys leu arg

Add T before “AAA”DNA-- TAC TTC TAA A CGC ATTmRNA 5’ AUG AAG AUU UGC GUA A 3’ met lys ile cys valCodons are shifted—bases that were in one codon are now a part

of another codon. Changes codon seq. Wrong aa are addedThrows off how ribo reads the mRNA: many aa subst.

Mut mRNA has one fewer base than WT mRNA due to base deletion

All codons from deletion site will be changed: mRNA is read differently

All codons from insertion site will be different: mRNA is read differently

Mutant mRNA has 1 extra base due to base insertion in DNA

• Chemicals– E.g. nitrous acid modifies bases and causes

substitution (A becomes G). Soot gets between bases and leads to insertion of extra bases in repl.

– Analogues: look like nucleotides; added to the new DNA strand during repl and causes substitutions

• Ionizing radiation– E.g. x-rays and gamma rays can break DNA strand;

breaks sugar-phosphate backbone

• Ultra violet light– T-T dimers stall replication and transcription

• DNA polymerase – makes mistakes at rate of 1/10E9 bases

Mutagens:Causes of Mutations

UV as Mutagen

• Exposure of DNA to UV results in the formation of covalent bonds b/w 2 adjacent thymines on same strand = thymine dimers

• When DNA containing thymine dimers undergoes replication, the following may occur:-- replication may not be completed (stoppage)-- new strand may have base subst-- may have base deletion-- may have base insertion

Replication

Stalling

base substitutions, base deletions or

insertions

UV as Mutagen

CATTG +UV= CAT-TG (red= thymine dimer)

DNA then replicates-done by DNA Pol

DNA Pol doesn’t recognize thymine dimer

- doesn’t know which bases to put into new strnd

- causes DNA Pol to stall at dimer; stops repl.

DNA Pol will try to “forge ahead” at times to complete replication- guess which bases to add

- DNA Pol will add bases at random to new strnd

- DNA Pol may also add wrong # bases to new strnd (1,3 or 4 bases instead of 2)

• Many repair mechanisms exist. E.g. excision repair

• DNA polymerase can fix its own mistakes (1/109 mutation rate)

DNA Repair

Figure 8.20

endonuclease

• Natural mutation rate with repair = 1 mistake per 109 bases

• 3’ to 5’ exonuclease activity: proofreading• E. coli chromosome has 4.6 x 106 bp= DNA

polymerase replicates chrmsme 217 times before it makes a single mistake

• Evolution? [infrequent] mistakes allow mutations accumulate in the genome over many generations

• May result in the production of new traits

The Frequency of Mutation

Bromo-uracil, an Analogue (chem mutagen)

BU – knock off A; looks like nuctide w/ base A

DNA repl in presence of BU, BU is placed into new DNA instead of A (BU goes where A is supposed to be)

GTCTA GTCTA-templ GTCTBU- new

CAGAT CBUGBUT-new CAGAT-templ

when BU is a part of DNA, it looks like a G. When DNA containing BU unergoes repl; Bu now looks like base G

GTCTBU GTCTBU-templ

CAGAT CAGAC-new results in base subst from t->c

CBUGBUT CBUGBUT-templ incorp of analogues results in

GTCTA GCCCA base sustitutions in dau DNA

Effect of Mutagens

• Cause mutations, or changes in the base sequence of DNA leads to changes in base sequence of mRNA

• May lead to changes in protein structure and function

• This may have harmful effects for the cell/organism

• Mutagens can cause cancers in humans (carcinogenic)

Identifying mutagens using bacteria

• Some chemicals are mutagens- carcinogenic in humans

• Need to id these chemicals so we can avoid them (and their harmful effects)

• Can use bacteria to help us identify mutagens• Find out if chemical can turn WT bact into MUT

bacteria• treat WT cells w/ chemical and see if the bacteria

can then grow on a selective media that allows ONLY mutant cells to grow

Identifying mutants

• Positive (direct) selection: detect mutants because they grow in selective media that allow only mutant cells to grow (ie- does NOT allow wildtype cells to grow)– E.g. can mutagen turn histidine-requiring WT

cells (his- ) into mutant cells that are histidine independent (his+ ) cells?

– Need to use WT cells whose growth properties you know well

Ames Test

• Type of positive selection• Divide WT cells into 2 batches: control and

experimental group• Control group- not exposed to chemical• Expt’al group- exposed to chemical; see if these

become mutant cells

The Ames Test is used in Positive Selection

Figure 8.22

Experimental groupWT Cells (his-) plus mutagenAnd liver extr (activator)

Control groupWT Cells plus liver extr only (no mutagen)

Compare # colonies B/w exptal (mutagenTreated gp) and ctrl(not mutagen treated)gp

Selective media:Allows only mutantHis+ cells to grow

If mutant group has More col, then suggestMutagen is creating Many mutants

In our example, WT cells need his in media to grow; MUT cells do NOT need his in media (can make own histidine)

Negative Selection requires Replica Plating

Identifies arg- mutants

argininearginine Mutant and WT can grow Only WT can grow

Mut cannot grow

Recombination –

• 2 DNA molecules exchange fragments with related/similar base sequences with each other

• Also refers to process by which one DNA molecule inserts into another DNA molecule

Recombination

Figure 8.23

Crossing over occurs b/w segments that have similar base sequences

Recombination: insertion

__a__b__c___d___e__f____ dna #1 D___E dna #2

__a__b__c___D___E__f___ dna #1 d___e dna #2 inserts into dna #1 dna more likely to insert itself into another

DNA at a region with a similar base sequence

Figure 8.2

Genetic transfer: onecell gives its geneticmaterial to another (unrelated) cell

Recombinant cell: has made donor DNA a part of its own chrosomsome (donor DNA has been inserted into recip. DNA)

Recombinant DNA: DNA molecule that contains DNA from 2 different organisms

Horizontal Genetic Transfer• Transfer of genetic

material/DNA from one bacteria to another bacteria of the SAME GENERATION

• Donor cell: cell whose DNA is being transferred [to another cell]-donor DNA: DNA of the donor cell; DNA which is transferred to another cell

• Recipient cell: the cell that gets/receives DNA from the donor cell

Horizontal Gene Transfer: how bacteria pick up new genetic material/info from other bacteria

3 Methods of Horizontal Gene Transfer

• Transformation – a recipient cell takes in naked donor DNA (DNA no longer contained in the donor cell) from its surroundings; in solution (eg-in liq media)

• Transduction – DNA is transferred from one cell to another by virus that infects bacteria (bacteriophage)

• Conjugation – transfer of plasmid from a donor bacterium to a recipient bacterium via the pilus-donor cell physically contacts recip using pilus

-plasmid travels thru pilus to get to recip. cell

Discovery of Transformation

Figure 8.24

Donor Cell

Dying donor cell: DNAis digested into fragments

Dead donor cell releases its DNA fragments (red) into its surroundings

Living cell in the vicinity ofreleased donor DNA frag takesin donor DNA frag (living cellIs the recip. cell)

Recip. Cell may insert donorDNA into its chromosome viarecombination: becomes recombinant cell

Recomb cell can now express gene contained in the integrated donor DNA and display the trait conferred by the geneIf donor DNA is not inserted into recip cell’s chromosome, it is degraded.

TRANSFORMATION

Recipient cell

Recombinant cell

Conjugation!!!

Conjugation: transfer of plasmid via pilus

Figure 8.27a

PLASMID

• Plasmid to be transferred must contain genes that code for pilus and other proteins that are involved in transfer process- having the plasmid gives bacteria the ability to do conjugation• Occurs only between a donor that has the plasmid (+ type) and a recipient that does not have the plasmid (- type)

ConjugationHfr transfer- formation Hfr cell

Figure 8.27b

Hfr transfer: conj done by bact that has inserted its plasmid into its chrmsme

Hfr cell: cell that has inserted plasmid into chrmsme

ConjugationHfr transfer

Figure 8.27c

Only the portion of the Hfr chromosomal DNA that has undergone replication will be transferred into the F- cell. Repl portion of chrmsme usually contains ONLY a small portion of plasmid seq; only a portion of the plasmid sequence is transferred to recip (- type) cell recip does NOT get the entire plasmid (recip considered F-)

Orange= plasmid integrated into chrmsome)

Replicated portionof hfr chromosome

Transduction

Figure 8.28

Recombinant

1

Phage protein coat

Bacterial chromosome

2

3

Bacterial DNA

Phage DNA

4Recipient cell

5

Donor bacterial

DNA

Recipient bacterial

DNA

Recombinant cell

A phage infects the donor bacterial cell.

Phage DNA and proteins are made, and the bacterial chromosome is broken down into pieces.

Occasionally during phage assembly, pieces of bacterial DNA are packaged in a phage capsid. Then the donor cell lyses and releases phage particles containing bacterial DNA.

A phage carrying bacterial DNA infects a new host cell, the recipient cell.

Recombinant can occur, producing a recombinant cell with a genotype different from both the donor and recipient cells.