Roles of Nucleotides in Cells.

MBIO 2100 

Constituents of DNA and RNA.



DNA:
Specifies sequence of every protein and RNA in cell.



Functions to store and transmit biological information.


RNA:
Broad range of functions.




rRNA
- components of ribosomes.




mRNA - carry genetic info from genes to ribosomes.




tRNA - translate info in mRNA into specific amino




acid sequence.

Lehninger Principles of Biochemistry, 4th ed.


CHAPTER 8

(Chapters 10, 3rd ed.)

Nucleotides and Nucleic Acids





All figures are from Lehninger 3rd ed. or 4th ed.

Hydrolysis of nucleoside triphosphates provides

chemical energy to drive a wide variety of
biochemical reactions.



ATP is most widely used. But GTP, CTP, and UTP are
used in some cases.

Nucleotides are structural components of enzyme

cofactors.

Energy currency in metabolic transactions. example?

Essential chemical links in response to hormones and

extracellular stimuli. example?

Structural components of enzyme cofactors.

Cells respond to their environment by responding to

hormones or other external chemical signals (first
messengers).

These external signals cause increases in second

messengers leading to metabolic changes inside cell.

Structure

Structure

Bases

Structure

Structure

base +

sugar +

phosphate

base +

sugar

Structure

Deoxyribonucleotides

General structures of nucleoside 5-mono-, di-, and triphosphates.

Structure

Ribonucleotides

Successive nucleotides in

DNA and RNA are linked

by phosphodiester bonds.



At pH 7 the phosphate

groups are completely ionized

and negatively charged.





The 5-end of the molecule

No nucleotide at the 5 position.

The 3-end of the molecule

No nucleotide at the 3 position.

RNA hydrolyzes rapidly under alkaline conditions.

Is DNA stable under alkaline conditions?

The nucleotide sequence of nucleic acids can be represented

schematically.



By convention, the 5-end of the nucleic acid is on the left,

and the 3-end is on right. 5> 3



Oligonucleotide = a short nucleic acid, < 50 nucleotides.

Polynucleotide = > 50 nucleotides.

Nucleotides and nucleic acids absorb UV light.

Properties of bases:

weakly basic

highly conjugated, resonance among ring atoms

planar (pyrimidines), nearly planar (purines)

absorb UV light

hydrophobic and relatively insoluble in water

Hydrogen bonds form between pairs of bases (A-T and G-C)

and permits complementary association of two nucleic acids
molecules

Chargaffs rules (1940s)

1. The base composition of DNA varies from one species to the
next.

2. DNA specimens from different tissues of the same species
have the same composition.



3. The base composition of DNA does not change with
organisms age, nutritional state, or changing environment.



4. In all cellular DNAs, the number of A = T, and G =C.

and A + G = T + C

(identify the types of bases: purine or pyrimidine?).

Structure of DNA:



Rosalind Franklin and
Maurice Wilkins, 1950s.



James Watson and Francis
Crick, 1953.



Two strands of DNA form a

double helix containing a

major groove (surco principal)
and a minor groove (surco
secundario).

The two strands of DNA in a double

helix are antiparallel and
complementary.

Three different forms of

DNA have been observed

experimentally.



B-form =

Watson-Crick form, most

stable for random sequence

DNA (right-handed)



Z-form = found in

short stretches in cells.

(left-handed)



A-form = occurs in cells?

(right-handed)

Watson and Crick suggested

that a DNA (blue) could be
replicated if its strands became
separated.



Each strand could serve as a

template for a complementary

daughter strand (red).

RNA



RNA found in nucleus and in cytoplasm. (DNA is

confined to the nucleus).



Increase in protein synthesis is accompanied by an increase in
the amount of cytoplasmic RNA.



mRNA carries genetic information from DNA to ribosome.
Nucleotide triplets code for amino acids.



TRANSCRIPTION = the process of forming mRNA on a DNA
template.



mRNAs transcribed from DNA are longer than the length
necessary to code for polypeptide sequence. The noncoding RNA
includes sequences that regulate protein synthesis.

The product of transcription of DNA is

always a single-stranded RNA.



Single-stranded RNA assumes a righthanded helical conformation. >

(bases = gray, phosphates = yellow, sugars = green)



When complementary sequences are

present, RNA forms an A-form helix.

B-form helices of RNA not observed.

Z-form helices of RNA observed in lab.

Unlike DNA, RNA does not form simple,

regular secondary structure.



Complex RNA structures are observed.



RNA can pair with other RNA, or with DNA.



Base pairing G-C, A-T is observed. Unusual
pairing, such as G-U is also observed.



Modified nucleotides are often present.

Base modifications and 3 base interactions !

RNAse P, functions in the
processing of tRNA

G pairs with U!

C-G-7mG

A - dmG pair

chemical properties

Duplex DNA is highly viscous at

room temperature, pH 7.



Extremes of temperature or pH:

H-bonds and base stacking
break.

Viscosity decreases, strands
separate DNA unwinds

(= denaturation).



DNA renatures in a rapid 1-step

process if conditions are
returned to lower temperature
or neutral pH (= annealing).

A-U-A

chemical properties

Duplex DNA has lower UV absorbance than single-stranded

DNA. Denaturation can be monitored by UV absorbance.

Denaturation is a cooperative process characterized by a
melting point, Tm.

Tm depends on the A+T and G+C content.

SEQUENCING (1977)

techniques: Maxam & Gilbert and Sanger.

Sanger sequencing:

template strand (to be sequenced)

primer

dNTPs

4 reactions, each with a different dideoxytriphosphates, ddNTP.

DNA polymerase



One of the dNTPs used is radioactive.

The fragments are separated according to size by electrophoresis.

Duplex DNA where denaturation has occurred in

in some regions (red arrows). These regions are likely

to be rich in A+T.

When a ddNTP is incorporated into the new chain, elongation of

the fragment can not proceed, because the 3-hydroxyl is
missing.

3-hydroxyl is not present in ddNTP.

Automatic DNA sequencing.

Chemical synthesis of DNA.

Sanger sequencing has been automated. The ddNTPs

are labeled with fluorescent tags.

MBIO 2100 




Lehninger Principles of Biochemistry, 4th ed.


CHAPTER 24

(Chapters 24, 3rd ed.)

Genes and Chromosomes:

Part 1





Figures and text are from Lehninger 3rd ed. or 4th ed.

1940s:

One gene-one enzyme.



Later:

One gene-one polypeptide.



Today:

Gene = a segment of DNA that encodes the
primary sequence of a polypeptide or RNA.

Minimum size for a polypeptide gene=

(# amino acid residues)(3 bp/residue)

How many genes in a chromosome?

E. Coli:

4,639,221 bp


4,300 proteins


115 RNAs

Humans:
3.2 x 109 bp




30,000 to 35,000 genes

BACTERIA

EUKARYOTES

genetic material is contained in
chromosomes.

diploid number (2n) depends on the
species



Example:

humans

DNA from a lysed E. coli cell.

Arrows indicate small circular
plasmid DNAs. >

E. Coli Genome:

double-stranded

circular

4,639,221 bp

in nucleoid.

24 pairs of different chromosomes

22 matched pairs, and


X + Y(male) or 2 X (female)

2 pairs of each chromosome, 46 total (diploid).

Example:

Wild yeasts

16 different chromosomes.

8 sets of chromosomes (octoploid).

Plasmids

replicate

pass to daughter cells at division.

Chromosomes of Nelson
or Cox

may pass from one bacterial cell to another.

some carry useful genes, for example resistance to antibiotics.

EUKARYOTES



organelles







Mitochondrial DNA (mtDNA)-

Circular duplex

< 20,000 bp in animal (bigger in plants)

2 to 10 copies per mitochondrion.

Code for mitochondrial rRNAs and tRNAs and for a few proteins.

95% mitochondrial proteins are encoded by nuclear DNA



Chloroplast DNA (cpDNA).

Circular duplex, 120,000 to 160,000 bp.

Most bacteria have

- one copy of genome


- one copy of each gene


- most genes colinear with protein sequences.



Eukaryotic genomes are more complex.

Note: some plants and some amphibians have more

base pairs of DNA in their genome than humans.



Viruses: 2,000 to 60,000 bp

Eukaryotic genes and chromosomes

are complex.





Eukaryotic genes contain
intervening sequences (=introns),
that separate the coding sequences
(=exons).
>



A few eukaryotic genes do not
contain introns (ex. histones).

Types of sequences in human genome.

What percent of the human genome

codes for proteins?



In humans:



exons are ~ 1.5% of the genome.

exons + introns ~ 30.% of the genome.

Transposons
(transposable elements) =
molecular parasites
within a host genome.
Some have genes encoding
proteins catalyzing
transposition.

Simple sequence repeats

(=SSR, satellite DNA)

Short, highly repetitive sequences
(< 10 bp)

Associated with centromeres and
telomeres.



Large segmental duplications (=SD)

Segments that appear more than
once in different locations.

A large proportion of the genome is

JUNK DNA

ENCODE:

Encyclopedia of DNA

Elements

A function can be

assigned to 80% of the genome.

http://www.nature.com/encode/

http://www.genome.gov/encode/

FIRST DRAFTS OF HUMAN

PROTEOME:

MAY 2014 IN NATURE



Catalog of proteins expressed
in non-diseased human tissues
and organs.

GENOME



TRANSCRIPTOME



PROTEOME



METABOLOME

How does all that DNA fit into the cell?

Cellular DNA is extremely compacted.

DNA molecules are much larger than the

cells that contain them.

example:

The human genome contains ~ 1

meter of DNA. Most cells are
diploid, and thus contain ~ 2
meters of DNA.

SUPERCOILING

(superenrollamiento)



Supercoiling = a coiled
coil.



Supercoiling is one of
the ways in which DNA is
compacted to fit into
cells.

SUPERCOILING



Supercoiling affects
replication and
transcription.



Supercoiling is highly
regulated in all cells.

Relaxed and supercoiled plasmids.

More relaxed
more supercoiled

<>



In circular DNA, supercoiling is maintained as long as the DNA
strands do not break.

In vivo DNA is underwound (< 1 vuelta/10.5 pb)

Underwinding the DNA makes it easier to separating
the two strands of DNA

Relaxed DNA:

1 turn/10.5 bp

10

B-form of DNA (= Watson-Crick structure)

The most stable form for random sequence DNA.



One turn of double helix per 10.5 bp.

(1 vuelta/10.5 pb)



DNA is relaxed. No supercoiling.



Supercoiling:

DNA underwound
< 1 vuelta/10.5 pb

DNA overwound

> 1 vuelta/10.5 pb

B-form of DNA (= Watson-Crick structure)

The most stable form for random sequence DNA.



One turn of double helix per 10.5 bp.

(1 vuelta/10.5 pb)



DNA is relaxed. No supercoiling.



Supercoiling:

DNA underwound
< 1 vuelta/10.5 pb

DNA overwound

> 1 vuelta/10.5 pb

Quantifying supercoiling:

In the cell the DNA is SUPERCOILED
and UNDERWOUND



DNA underwound
< 1 vuelta/10.5 pb

Linking Number, Lk =





(#bp )(1 turn/10.5 bp)

Quantifying supercoiling: Lk = (#bp )(1 turn/10.5 bp)

What is the Linking Number (Lk) of a relaxed,
circular molecule of DNA containing 5,250
base pairs?

Lk
= #bp (1 turn/10.5 bp)


= 5,250 bp (1 turn/10.5 bp)


= 500 turns

relaxed

example

DNA relaxed: (2,100 bp)(1 turn/10.5 bp) = Lk0 = 200.

Remove 2 turns (DNA underwound): Lk = 198 turns.

Lk = Lk - Lk0

11

Quantifying supercoiling: Lk = #bp (1 turn/10.5 bp)

example:


DNA relaxed: (2,100 bp)(1 turn/10.5 bp) = Lk0 = 200.


Remove 2 turns (DNA underwound): Lk = 198.

Lk = Lk - Lk0 = 198 - 200 = -2

Quantifying supercoiling: Lk = #bp (1 turn/10.5 bp)

example:


DNA relaxed: (2,100 bp)(1 turn/10.5 bp) = Lk0 = 200.


Remove 2 turns (DNA underwound): Lk = 198.

Lk = Lk - Lk0 = 198 - 200 = -2

Specific linking difference = = Lk/Lk0

: is independent of the length of the DNA molecule

example:


Lk/Lk0 = -2/200 = -0.01
-> the DNA is 1% underwound

: is independent of the length of the DNA molecule

example:


Lk/Lk0 = -2/200 = -0.01
-> the DNA is 1% underwound

negative = fewer turns than relaxed DNA

Cellular DNAs are usually

5%7% underwound

( = -.05 to -.07)





Underwinding = fewer helical
turns than found in the B-form
of DNA. Produces negative
supercoils.



Overwinding = more helical
turns than found in the B-form
of DNA. Produces positive
supercoils.

Specific linking difference = = Lk/Lk0

:


is independent of the length of the DNA molecule

Topoisomerases = Enzymes that increase or

decrease the amount of DNA underwinding.

Topoisomerases change the linking number

(Lk).



Play important roles in replication and
DNA packaging.

Experiment: Agarose gel

electrophoresis.





Highly supercoiled DNA

more compact than relaxed DNA

therefore migrates more rapidly.

Lane 1:

supercoiled and relaxed plasmid

Lanes 2 and 3:

incubate with topoisomerase

DNA in chromatin is tightly associated with small proteins called

histones. Many other nonhistone proteins are also found in
chromatin.



DNA and histones form nucleosomes.



The nucleosome are arranged like beads on a string (perlas en un
collar).

12

The binding of histone cores to

DNA is not random.



Exactly where the histone binds
is not yet understood, but it
seems to depend on the
presence of AT basepairs in the
DNA.


DNA wrapped around a nucleosome core.

Nucleosome core contains 8 histones.



146 bp of DNA are wrapped around the core, 54 bp of DNA
forms linker between nucleosomes.

Nucleosome cores are arranged into a structure called

the 30 nm fiber. This structure causes DNA to be ~ 100X
more compact.



Not all the DNA in chromosomes is arranged in 30 nm fibers.



The presence of 30 nm fibers appears to depend on
transcriptional activity.

Higher levels of organization exist but they are not

well understood.

Certain regions of the DNA associate with the
nuclear scaffold.

The loops often contain related genes.

Histone modifications (acetylation, methylation,

etc.) appear to determine which genes are active.

Histone Code



PMID 18250624

Nat Rev Genet. 2008;9:179-91.

Genome-wide approaches to studying chromatin
modifications.

Schones DE, Zhao K.

Additional layers of organization exist,

each makes the DNA more compact.



The higher levels of organization are
not well understood.



Proposed model for further levels of
organization >

13