DNA dan RNA. 1. Lehninger. Principles of Biochemistry. 4th Edition. 9/20/2016

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DNA dan RNA

• Struktur fisik dan kimia DNA,

• Orientasi antiparalel dsDNA, tipe pilinan untai DNA, • Denaturasi, kurva leleh dan renaturasi,

• Sekuen basa dan konsensusnya

1. Lehninger. Principles of Biochemistry. 4th

Edition.

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• The component parts of DNA – Nitrogenous bases: • Adenine (A) • Cytosine (C) • Guanine (G) • Thymine (T) – Phosphoric acid – Deoxyribose sugar – Nitrogenous bases

• Like DNA except Uracil (U) replaces Thymine – Phosphoric acid – Ribose sugar N u c le o s id e s la ck th e p h o sp h o ri c a ci d

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• Nucleotidesare nucleosides with a phosphate group

attached through a phosphodiester bond

• Nucleotides may contain one, two, or even three phosphate groups linked in a chain

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Summary

• DNA and RNA adalah rantai molekul yang

tersusun atas subunit nukleotida

• Nukleotida terdiri dari basa nitrogen yang

berikatan dengan gula dan gugus fosfat

• Gugus fosfat berikatan dengan gula DNA atau

RNA melalui ikatan fosfodiester ujung 5’ dan

ujung 3’

BACKBONE

• The backbones of both DNA and RNA are hydrophilic. The hydroxyl groups of the sugarresidues form hydrogen bonds with water.

• The phosphate groups are completely ionized and negatively charged at pH 7, and the negative chargesare generally neutralized by ionic interactions with

positive charges on proteins, metal ions, and polyamines.

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NITROGEN BASE

• The purine and pyrimidine bases are hydrophobic and relatively insolublein water at the near-neutral pH of the cell.

• At acidic or alkaline pH the bases become charged and their solubility in water increases.

NITROGEN BASE

• Free pyrimidines and purines are

weakly basic compounds and are thus called bases.

• They have a property with important

consequences for the structure, electron distribution, and light absorption of nucleic acids. • Other result is that all nucleotide

bases absorb UV light, and nucleic acids are characterized by a strong absorption at wavelengths near 260 nm

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DNA Structure

The

Double Helix

• Rosalind Franklin’s x-ray data suggested that DNA had a helical shape

• The data also indicated a regular, repeating structure • DNA was believed to require an irregular sequence • Watson and Crick proposed a double helix with

sugar-phosphate backbones on the outside and bases aligned to the interior

DNA Helix

• Structure compared

to a twisted ladder

– Curving sides of the ladder represent the sugar-phosphate backbone

– Ladder rungs are the base pairs

– There are about 10 base pairs per turn

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MAJOR & MINOR GROOVE DNA

• Untaian DNA mempunyai dua lekukan

(groove) eksternal:

- Lekukan besar/ major groove

- Lekukan kecil/ minor groove

• Fungsi groove: tempat lekatan beberapa

protein

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Summary

• The DNA molecule is a double helix, with

sugar-phosphate backbones on the

outside and base pairs on the inside

• The bases pair in a specific way:

– Adenine (A) with thymine (T)

– Guanine (G) with cytosine (C)

Bentuk fisik kimia nukleotida

DNA dan RNA dapat mempunyai bentuk

yang bervariasi

– Perubahan

kelembaban selular

relatif dapat

menyebabkan perubahan molekul DNA

– DNA umumnya berputar kearah

right-handed

(A-form dan B-form) tetapi arah

left-handed

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A Variety of DNA Structures

• Ketika kelemban tinggi, DNA membentukB-form

• Kelembaban rendah sekitar 75%, membentukA-form

Tipe untaian Base pair/

putaran Rotasi/ base pair (°) untaian (°A)Diameter _KelembapanKondisi

relatif (%) Dalam larutan

A 11 +32,7 23 75 K+, Na+

B 10 +36 19 92 Kadar garam rendah

Z 12 -30 18 - Kadar garam

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Variation in DNA between Organisms

• Ratios of G-C and A-T are fixed in any specific organism

• The total percentage of G+C varies over a range to 22 to 73%

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DNA Denaturation

• In addition to heat, DNA

can be denatured by:

– Organic solvents – High pH

– Low salt concentration

• GC content also affects

DNA density

– Direct, linear relationship – Due to larger molar volume

of an A-T base pair than a G-C base pair

• Dengan pemanasan, ikatan nonkovalen DNA menjadi lemah dan lepas

• Ketika lepas, 2 benang DNA akan terpisah yang disebut

denaturationataumelting

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DNA Renaturation

• After two DNA strands separate, under proper

conditions the strands can come back together

• Process is called

annealing

or

renaturation

• Three most important factors:

– Temperature – best at about 25 C below T_m – DNA Concentration – within limits higher

concentration better likelihood that 2 complementary will find each other

– Renaturation Time – as increase time, more annealing will occur

Polynucleotide Chain Hybridization

Hybridization is a process of

putting together a

combination of two different

nucleic acids

– Strands could be 1 DNA and 1 RNA

– Also could be 2 DNA with complementary or nearly

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Summary

• GC content of a natural DNA can vary from less

than 25% to almost 75%

• GC content has a strong effect on physical

properties that increase linearly with GC content

– Melting temperature, the temperature at which the two strands are half-dissociated or denatured – Density

– Low ionic strength, high pH and organic solvents also promote DNA denaturation

Sekuen Basa

dan

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DNA Sizes

DNA size is expressed in 3 different ways:

– Number of base pairs

– Molecular weight – 660 is molecular weight of

1 base pair

– Length – 33.2 Å per helical turn of 10.4 base

pairs

Measure DNA size either using electron

microscopy or gel electrophoresis

Relationship between DNA Size and Genetic Capacity

How does one know how many genes are in

a particular piece of DNA?

– Can’t determine from DNA size alone

– Factors include:

• How DNA is devoted to genes? • What is the space between genes?

– Can estimate the upper limit of number genes

a piece of DNA can hold

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Summary

• Natural DNAs come in sizes ranging from

several kilobases to thousands of

megabases

• The size of a small DNA can be estimated

by electron microscopy

• This technique can also reveal whether a

DNA is circular or linear and whether it is

supercoiled

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DNA Size and Genetic Capacity

How many genes are in a piece of DNA?

– Start with basic assumptions

• Gene encodes protein

• Protein is abut 40,000 D

– How many amino acids does this represent?

• Average mass of an amino acid is about 110 D • Average protein – 40,000 / 110 = 364 amino acids • Each amino acid = 3 DNA base pairs

• 364 amino acids requires 1092 base pairs

DNA Genetic Capacity

How large is an average piece of DNA?

–

E. coli

chromosome

• 4.6 x 106_bp

• ~4200 proteins

– Phage

l

(infects

E. coli

)

• 4.85 x 104_bp

• ~44 proteins

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DNA Content and the C-Value Paradox

• C-value is the DNA content per haploid

cell

• Might expect that more complex

organisms need more genes than simple

organisms

• For the mouse or human compared to

yeast this is correct

• Yet the frog has 7 times more per cell than

humans

C-Value Paradox

• The observation that more complex

organisms will not always need more

genes than simple organisms is called the

C-value paradox

• Most likely explanation for the paradox is

that

DNA that does not code for genes is

present

when the less complex organism

has more DNA

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Summary

• There is a rough correlation between DNA

content and number of genes in a cell or

virus

• This correlation breaks down in several

cases of closely related organisms where

the DNA content per haploid cell (C-value)

varies widely

• C-value paradox is probably explained not

by extra genes, but by extra noncoding

DNA in some organisms

RNA

Basa yang ditemukan pada nukleotida adalah basa purin (adenin = A, guanin = G) dan basa pirimidin (cytosin = C, urasil = U)

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• Struktur RNA



untai tunggal sehingga tidak

membentuk struktur heliks yang teratur seperti DNA.

• RNA bisa membentuk struktur sekunder dan tersier

karena pasangan basa bisa terbentuk pada daerah yang

membentuk loops.

• Terdapat tiga tipe molekul RNA dalam sel, yaitu:

– mRNA



messenger

RNA

– tRNA



transfer

RNA

– rRNA



ribosomal

RNA

 Messenger RNA (mRNA) merupakan blue print yang

mengkode urutan asam amino dari satu atau lebih polipeptida yang terdapat dalam satu gen atau sekumpulan gen.

 Hasil transkripsi gen:

◦ organisme prokariot dapat langsung ditranslasi menjadi protein

◦ organisme eukariot harus melalui proses tambahan untuk menghilangkan intron.

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 mRNA eukaryotik umumnya bersifat monogenik, sedangkan mRNA prokaryotik bersifat poligenik

 MonogenikmRNA membawa sandi untuk satu polipeptida

 Ujung 3’ pada sebagian besar mRNA eukaryotik memiliki sebuah ekor terdiri dari 100-200 residu A berurutanekor poli A

 Kepala 5’ mRNA eukaryotik yaitu suatu

7-metilguanosinyang berikatan trifosfat dengan residu terminal 5’

 The 5 cap helps protect mRNA from ribonucleases

tRNA (transfer RNA)



75-95 nukliotida



± terdapat 61 jenis tRNA

di sitoplasma yang membawa asam amino

yang berbeda



membawa asam amino dari sitoplasma ke

ribosom dalam proses translasi kodon

mRNA menjadi rangkaian asam amino yang

kemudian membentuk protein

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Struktur tRNA

 struktur tRNA berbentuk seperti daun (cloverleaf).

 Pada tRNA terdapat urutan tiga basa yang disebut antikodon.  Antikodon ini komplemen dengan

salah satu kodon.

 ujung 3’ tRNA terikat asam amino spesifik.

 tRNA yang sudah mengikat asam amino disebut aminoasil tRNA.

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Processing of tRNAs in bacteria and eukaryotes

CCA is then added to the 3 end, a necessary step in processing eukaryotic tRNAs and those bacterial tRNAs that lack this sequence in the primary transcript. The 3-terminal trinucleotide CCA(3) to which an amino acid will be attached during protein synthesis. For the eukaryotic tRNA, the final step is splicing of the 14-nucleotide intron. Introns are found in some eukaryotic tRNAs but not in bacterial tRNAs.

• The primary transcripts of prokaryotic and eukaryotic tRNAs are processed by the removal of sequences from each end (cleavage) and in a few cases by the removal of introns (splicing).

• Many bases and sugars in tRNAs are also modified; mature tRNAs are replete with unusual bases not found in other nucleic acids

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rRNA

• Merupakan molekul RNA penyusun sub unit ribosom

• Struktur rRNA mengandung banyak loops, dan terdapat pasangan basa di antara loops

• rRNA memiliki pasangan basa internal yang membentuk kompleks dengan protein membentuk partikel ribonu-kleoprotein yang disebut ribosom

• rRNA pada prokariot5S, 16S dan 23S rRNA

• rRNA pada eukariot5S, 18S dan 28S rRNA

• Pada prokariot subunit kecil mempunyai ukuran 30S dan subunit besar 50S, sementara pada eukariot subunit kecil 40S dan subunit besar 60S.

16 S rRNA