I] DNA and RNA



Molecular Genetics

T.A. Blakelock High School Grade 12 University Preparation Biology

Student:______________________ Date:___________________ Teacher:______________________ Room:__________________ Period:_________

Unit 3 MOLECULAR GENETICS

INTRODUCTION

Pg 198 ‘ARE YOU READY?’

Part I DNA, RNA and PROTEIN SYNTHESIS

A] HISTORY OF DNA Figure 11, pg 215

NOTABLE SCIENTISTS:

____________________________ : Discovery that the Nucleus is the source of inheritance

______________________ and __________________ : Showed that DNA actually controls heredity

____________________________ : Crystallography Structure of DNA

____________________ and _____________________ : Mathematical modeling of DNA Structure

B] DNA STRUCTURE

1] Parts of a nucleotide

____________________ _________________ ______________________

2] arrangement of nucleotides

a] the __________ bonds to the last _______________ at carbon _____

and so the sugar end of DNA is called the _______ end

b] the ______________ bonds to the next ___________ at carbon ____

and so the phosphate end of DNA is called the _____ end

c] the two strands are arranged as shown below

Sections of the DNA with more ________________ bonds, are more stable.

[_____ H-bonds vs. ______ H-bonds]

4. DNA shape – the stands are arranged in a ________________________________

Each Human Cell contains approximately ___________________________ base pairs.

These are arranged into roughly ______________________ different genes.

In addition, up to __________________ does not code for any protein.

In total, DNA from 1 cell could be _________________ long.

___________________ is very stable but ______________ is very easily __________________.

C. DNA REPLICATION

Introduction

a. Before a cell _________________ it must first copy all its DNA

b. It must do this while keeping the amount of ______________ to a minimum.

c. Also, the DNA will have to be __________________ to allow access to the bases.

[The bold numbers following the bold terms refer to the DNA picture of replication on the next page]

1. Separating the strands

a] DNA helicase [1] – breaks___________ between the strands at an “origin of replication”

b] DNA topoisomerase [2] - releases _____________ by unwinding

c] replication fork [3] - an area where the two strands are _________________

d] ssB’s [4] - single-stranded binding proteins keep strands from_______________

2. Building complementary strands

a] starting -DNA primase [5] - creates a _____________ strand of RNA

called an RNA-primer[6] at the exposed______end of the replication fork

b] building -the enzyme DNA polymerase III [7]- adds complimentary bases that are

_________________ moving along the old DNA in the ________ direction

- the energy released by removing ______________ allows the bonding

- the strand that is copied continuously __________ strand is

called the leading strand [8]

c] other strand DNA polymerase III only moves__________________

- so the lagging strand [9] has to be built_________________________

- these ____________________ pieces are called Okazaki fragments [10]

d] finishing DNA polymerase I [11] removes the __________________________

& replaces them with the appropriate DNA bases

DNA ligase [12] attaches __________________ fragments

by bonding sugar and ___________________________

3. Picture – see next page

___ DNA Helicase -

___ DNA Topoisomerase -

___ DNA Ligase -

___ DNA Polymerase I -

___ DNA Polymerase III -

___ Lagging strand -

___ Leading strand -

___ Okazaki fragments -

___ DNA Primase -

___ Replication Fork -

___ RNA primer

___ ssB’s -

D. RNA

5. RNA – ribonucleic acid

RNA Structure

a] _________ sugar + phosphate + nitrogenous base but ____________ replaces Thymine

b] __________ stranded molecule

c] much _______________ than DNA

5’ P—sugar—P—sugar—P—sugar—P—sugar—P—sugar—P—sugar 3’

l l l l l l

A C G C U G

RNA features

unlike DNA, RNA can _______________ in the cytoplasm if properly prepared

Types of RNA

1. mRNA – messenger RNA

- varies in _______________, but always much _______________ than DNA

- carries the ______________ message for protein building from DNA to the _____________

2. tRNA – transfer RNA

- very short, _________________ bases only

- acts to transport the ________________________ to the mRNA at the ribosomes

3. rRNA – ribosomal RNA

- varies in length, but ______________ than mRNA

- binds the large and small __________________ to form a functional _________________

E. TRANSCRIPTION – the process of making mRNA

1. Initiation

a. ______________________ binds to a specific region of the DNA

that is _______ rich and so easy to separate

b. the DNA is _________________ and the two strands are _________________

2. Elongation

a. only the ___________________ DNA strand is used ( _________________ strand

b. the unused strand [5’ to 3’] is called the_________________ strand

c. free floating nucleotides A, U, C & G are __________________

to their complimentary bases by ______________________

d. RNA polymerase moves along the DNA _________________& _______________

as it goes while the DNA winds back up behind it

e. the ____________ hangs free off the side of the DNA strand

attached only at a few base pairs

3. Termination

a. RNA polymerase encounters a sequence of bases at the end of the gene

called a ___________________________________

b. this results in RNA polymerase and the _______________ being released from the DNA

4. Post-transcriptional modifications

a. a ______________ is added to the mRNA to protect it from cytoplasmic enzymes

b. a ________________ of 200 or so adenines is added by poly-A-polymerase to the 3’ end

c. this results in the _____________________ being ready for release from the nucleus

d. in eukaryotes__________________ remove all the non-coding regions [_____________]

and ____________ the coding regions [_________________]

to form the ____________________________________

F. PROTEIN SYNTHESIS [or DNA translation] converting the RNA message into protein

1. The code mRNA

a. the message on mRNA is written in 3-letter ____________ called ______________

b. there are ________ or ____________ possible codons

c. pg 248 shows the codons – you will have to learn the ______________

but NOT memorize this with the exception of the __________ and the ___________ codons

d. notice that UUU & UUC both code for _____ and CUU, CUC, CUA & CUG all code for_____

this indicates that the __________ letter is the least important in the code

e. only two amino acids have a unique code UGG ( _______ and AUG ( _________

f. all proteins must start with ________ as AUG is the __________code; this may be removed later

g. there is__________ amino acid that is coded by UAA, UAG or UGA

and so the protein breaks here and these are called _______________codes

2. The ribosomes [rRNA]

a. the rRNA holds together the ____________ subunit [60s] and the___________ subunit [40s]

to form the active ribosome [___________]

b. the _________ [CAP region] adheres to the active ribosome

c. the ribosome will move the _________ through it

starting at the _________ end and going toward the _________ end

3. The tRNA

a. each tRNA has a_________________________ structure [pg 251]

b. at the base there is an _______________ which is the compliment of the _____________ on

the mRNA

eg the mRNA has the codon ________ for met ( the tRNA—met has the anticodon ______

c. at the 3’ end of the tRNA is the _______________that holds the amino acid

d. it requires 1 ATP to attach each amino acid to the correct tRNA to make an ________________

e. there are ______ possible tRNA for the 20 aa, therefore a cell needs ____ enzymes for this

4. Protein Synthesis

STEPS IN PROTEIN SYNTHESIS

E-site P-site A-site

From the piece of DNA below, make the complimentary strand of DNA, transcribe the appropriate strand, package it for the cytoplasm and then translate it as described in the steps below.

In row A; make the complimentary DNA strand; In row C, transcribe the DNA into RNA;

In column 1 and 28, place the appropriate 3’ or 5’; In column 2 & 27, ‘package’ the RNA in row C;

In row D, underline the ‘words’ in the RNA; In row E, translate the RNA into amino acids using the 3-letter codes for them

| |1 |2 |

| | | |

|Genus | | |

|species | | |

|strain | | |

|isolate | | |

C. Methylases

a. These enzymes add –_____________ groups to the nitrogenase bases

b. A methylated base will __________________ be recognized by a restriction enzyme

& the DNA will NOT be _________________

c. This allows researchers to ______________ sections of DNA before using the restriction

enzymes

DNA Ligase

d. This enzyme attaches ssDNA _______________________________________

e. After, the ‘sticky ends’ form _______________________________ to hold the bases together

eg.

D. Gel Electrophoresis

a. a procedure for _____________ the cut pieces of DNA to allow researchers to select the one they want

b. a gel is a _____________________ substance made of starch, agarose or polyacrylamide

c. electrophoresis refers to separation based on _________________________

d. ______________ pieces of DNA will move further than large pieces of DNA – separation by________

e. so that researchers can find the DNA on the gel – ___________________________ is added

this chemical fits into the twists of DNA without altering it and __________________ under UV light

f. DNA pieces can now be cut, separated & isolated from the gel and checked for the

___________ gene

Using DNA fingerprinting [which does not involve _________________________]

1. Paternity tests

the child’s DNA has to match both __________________ and _________________

2. Criminal investigations

eg. ________________ where the victim and the ___________ are both sampled

eg. ________________ where matching the _____________ is the key

6. Polymerase chain reactions

make notes from web presentation D.

Increasing the DNA ( Polymerase Chain Reactions [PCR]

[Refer to Youtube -NOT ON POWERPOINT]

Why do researchers do this?

They wish to amplify _______ copies of DNA to ____________ copies.

What section of DNA is amplified?

Only a _______________ section of the DNA is amplified.

It is chosen because it is highly ____________________ and is called the ___________ DNA.

The idea is that if we look at __________________ of these sections, any one person should have a

________________ pattern.

Procedure:

Start with the sample of DNA and add:

1. _____________________: short [20 nucleotide] pieces of DNA of known sequence that

will bond to both the _____________ and the _________________ strands.

2. _____________________: the basic building blocks of DNA

3. _____________________: an enzyme from a thermophilic bacteria that lives in

_______________ and so is adapted to very warm temperatures.

Cycles:

a. heat to _____oC: The heat _____________ the DNA breaking ___________ bonds.

b. cool to _____oC: To allow the primers to _____________ to complimentary sequences on both of the strands.

c. heat to _______oC: To allows Taq polymerase to _______________ DNA by adding

complimentary bases.

Results:

one cycle ( _______ copies

two cycles ( _______ copies

three cycles ( _______ copies [general formula ________________________]

twenty-five cycles ( __________________ copies

The amplified sample is now run on a ____________ to separate the DNA pieces based on size and

to establish the _____________ identity of the sample.

GENETIC ENGINEERING

A. Overview

1. _________________ the DNA segment with the desired gene

2. _________________ the DNA segment from the genome

3. __________________________ to place the desired gene in a new organism

B. Finding the DNA segment

1. Based on starting with a __________________

a. -make a synthetic ___________________ based on the amino acid sequence of the protein

b -allow a radioactively labeled mRNA to __________________

to heated, single-stranded DNA [ssDNA]

c. – the labeled mRNA will stick to piece of DNA with the_______________ gene

2. Based on starting with _________________

a. –find a cell that is active in synthesis of the protein so that it’s making lots of ____________

b. –isolate the mRNA from the cytoplasm and label it radioactively then allow it

to __________________ to heated ssDNA as above

6 if the piece of labeled mRNA anneals to piece 6, 7 as well as 8 it may be because of exons

and so a different restriction enzyme would have to be used

STEPS TO SOLVING LOCATION of GENE PROBLEM

STEP 1 – Write the segment numbers of the digested DNA on the samples of the gel grid, based on

relative lengths of the segments.

STEP 2 – Based on step 1, write the segment letters on the digested DNA grid corresponding to the

few samples for which protein synthesis data was obtained.

STEP 3 – Identify the segment of DNA that produces a normal protein. This segment contains the

gene, and likely more DNA on both ends, the next steps will allow you to eliminate

the excess DNA that is not part of the gene.

STEP 4 – Any segment that produces a smaller, not useful, protein is missing the stop codon at the

end of the gene.

STEP 5 – Any segment that produces no protein until a promoter is added, has the start codon, but is

missing the promoter that is found just upstream from the gene.

STEP 6 – The final segment of DNA that is actually needed should include the shortest segment that

contains the stop codon and the start codon plus a tiny upstream segment.

A] Samples of identical DNA digested by restriction enzymes

B] Gel electrophoresis samples

|EcoR I |Hind III |Sol I |

| | | |

• Band ‘C’ made a normal protein.

• Band ‘F’ made a smaller protein that is not useful.

• No Sol I band made a protein, but when a promoter was added,

band ‘M’ did make a normal protein.

On the grid at the top, show where the gene is located, showing your “calculations” on the gel grid, the digested DNA and on a separate page if necessary.

eg.

D. Vectors – to get the gene into other cells

1. Plasmids

a. these are small circular pieces of _____________ found in bacteria

b. bacteria have restriction enzymes to allow them to cut their own large ______________________

and integrate ___________________ into it

c. most _____________________ resistance genes occur on plasmids

d. there are genetically engineered plasmids that have known restriction enzyme __________________

and known antibiotic ________________________

e. the _______________ [segment of DNA selected from the electrophoresis procedure and annealing

with the mRNA] is now placed into the _____________________ using the restriction enzymes

f. the plasmid is now placed into bacteria and if the bacteria ____________________ the plasmid

1( it will have the _________________________

2 ( it will resist the ________________________

g. researchers screen _________________ of bacteria to find the few that have integrated the plasmid

h. however, without the _______________________ region, the target gene will not work in the bacteria

i. these plasmids may also be used to infect _________ and integrate the target gene into the _______

2. Viruses

a. a virus is used as a ___________________ instead of a plasmid

b. the virus is modified to remove all of the ___________________ genes, if possible

c. these must be _______________ viruses that undergo a _____________________ cycle

d. the virus is then used to infect ____________________ cells

and introduce the target gene into the __________________________ cell

-----------------------

O

II

HO—P—OH

I

OH

____ end

l

base—sugar

l

P

l

base—sugar

l

P

l

base—sugar

l

P

l

____ end

_____ end

l

P

l

sugar—base……

l

P

l

sugar—base……

l

P

l

sugar—base……

l

_____ end

pyrimadine

___________

___________

purine

___________

___________

O

OH

CH2

5

3

3

5

3

12

11

10

9

8

7

7

7

6

O

OH

CH2

5’

3’

3’

5’

3

12

11

10

9

8

7

7

7

6

5

4

2

1

DNA Replication

3’

5’

5’

3’

3’

5’

5’

3’

5’

genes are_________

three β-galactosidase genes

lacA

lacY

lacZ

OPERATOR

PROMOTER

genes are ______________

three β-galactosidase genes

lacA

lacY

lacZ

OPERATOR

PROMOTER

EXON

INTRON

EXON

INTRON

EXON

INTRON

EXON

INTRON

EXON

INTRON

EXON

INTRON

UAC

Met

G C C T A A T C G T C A C T G C A A

C T

3’—C G C A T T G C G T A A C G—5’

5’—G C G T A A C G C A T T G C—3’

G A

C G G A T T A G C A G T G A C G T T

G T G C A C

C A C G T G

HOH2C

OH

OH

O

O

II

HO—P—OH

I

O

combined to form

______________

UAC

CC GAG CUC AUG CCA AUC GGC CAG GUA UAG GGG ACC

E-site P-site A-site

Val

CAU

Gln

GUC

Gly

CCG

Ile

UAG

Pro

GGU

Met

UAC

CC GAG CUC AUG CCA AUC GGC CAG GUA UAG GGG ACC

4. The tRNA with ____________ attached, randomly enters the _____________ on the ribosome.

Because its ________________ matches the _____________ in the P-site, this tRNA is held in place.

P-site A-site

CC GAG CUC AUG CCA AUC GGC CAG GUA UAG GGG ACC

Val

CAU

Gln

GUC

Gly

CCG

Ile

UAG

Met

UAC

Pro

GGU

_______ subunit of ribosome

2. The mRNA ____________ the nucleus and encounters the _______________ of the ribosome.

3. The________________ attaches the large subunit to the small subunit that is attached to the mRNA.

The tRNA’s are ____________ by the attachment of an amino acid, by the use of an _____________.

CC GAG CUC AUG CCA AUC GGC CAG GUA UAG GGG ACC

______ subunit of ribosome

UAC

CC GAG CUC AUG CCA AUC GGC CAG GUA UAG GGG ACC

1. The mRNA, rRNA and tRNA are ____________ from DNA in the ___________. They randomly drift around

in the _______________where the large and small subunits of the _____________ are also drifting.

3. Hydrogen bonding

- adenine forms _____ hydrogen bonds with thymine

- cytosine forms _____ hydrogen bonds with guanine

Since A – T and C – G are the usual bondings,

if the amount of A does not equal T or the amount of

C does not equal G, then the DNA is not

double stranded

Mary Bob Larry Child

L

M

N

O

P

F

G

H

I

J

K

A

B

C

D

E

1 2 3 4 5

12 13 14 15 16

6 7 8 9 10 11

EcoR I

Hind III

Sol I

exon 1 exon 2 exon 3

segment segment segment

6 7 8

Locating a complete gene on a segment of DNA

5 the mRNA ________ to

the piece with the

target gene sequence

3 heat the DNA

to make it

________

+

4 add a piece of _______

mRNA for the

desired gene

2

1

cut with ______________ enzymes

separated after being run on the

_____________gel

8

5

6

3

9

10

2

1

4

7

1 2 3 4 5 6 7 8 9 10

protected DNA after being cut and first 2 segments ligated

CH3

CH3

protected DNA [red] cut with restriction enzymes

unprotected DNA cut with restriction enzymes

uncut DNA

Hind III

EcoR1

Sal 1

A A G C T T

T T C G A A

G A A T T C

C T T A A G

Met-Pro-Ile-Gly-Gln-Val

CAU

CC GAG CUC AUG CCA AUC GGC CAG GUA UAG GGG ACC

UAG

6. The tRNA that brought Met now

is ____________ after Met is _________ to

the Pro. The ______ is now moved through

the ribosome until the ___________ with

Met-Pro is in the ________. The tRNA for

the next code now fits into the _______

P-site A-site

UAC

Ile

UAG

Gln

GUC

Gly

CCG

Val

CAU

Met-Pro

GGU

CC GAG CUC AUG CCA AUC GGC CAG GUA UAG GGG ACC

5. The tRNA for the next

amino acid _______________

moves into the ______________

P-site A-site

Val

CAU

Gln

GUC

Gly

CCG

Ile

UAG

Pro

GGU

GGU

CCG

GUC

7. The processes of step 5 & step 6

are repeated until one of the ________

codes is in the _____________.

UA UAG GGG ACC

CCA AUC GGC CAG G

________________

Met-Pro-Ile-Gly-Gln-Val

CC GAG CUC AUG CC

8. Because there was no tRNA to

match _________________, the

polypeptide chain is ____________

and the ribosome ______________.

The mRNA will be _____________.

At this point all of the amino acids have been bonded into a________________

chain conected to the last tRNA in the _______________.

E-site P-site A-site

E-site P-site A-site

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