9/20/2016
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. 2.
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Nucleosides lack the phosphoric acid
• RNA component parts – Nitrogenous bases • Like DNA except Uracil (U) replaces Thymine – Phosphoric acid – Ribose sugar
• The component parts of DNA – Nitrogenous bases: • Adenine (A) • Cytosine (C) • Guanine (G) • Thymine (T) – Phosphoric acid – Deoxyribose sugar
Purines and Pyrimidines
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• Nucleotides are 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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Synthesis is always 5’ to 3’
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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 sugar residues form hydrogen bonds with water. • The phosphate groups are completely ionized and negatively charged at pH 7, and the negative charges are 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 insoluble in 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.
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They have a property with important consequences for the structure, electron distribution, and light absorption of nucleic acids.
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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 sugarphosphate 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
• Arrows indicate that the two strands are antiparallel
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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 (Z-form) ditemukan pada 1979
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A Variety of DNA Structures • Ketika kelemban tinggi, DNA membentuk B-form • Kelembaban rendah sekitar 75%, membentuk A-form
Tipe untaian
Base pair/ putaran
Rotasi/ base pair (°)
Diameter untaian (°A)
Kondisi
A
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+32,7
23
75
K+, Na+
B
10
+36
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92
Kadar garam rendah
Z
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-30
18
-
Kadar garam tinggi
Kelembapan relatif (%)
Dalam larutan
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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% • Such differences are reflected in differences in physical properties
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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 denaturation atau melting Suhu dimana ½ DNA strands terdenaturasi di sebut melting temperature or Tm Kandungan G-C content berpengaruh terhadap Tm . Semakin tinggi G-C semakin tinggi pula Tm
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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 Tm – 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 complementary sequences
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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 Konsensusnya
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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
– Phage x174 (one of smallest) • 5375 bp • ~5 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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RNA • 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
mRNA 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-metilguanosin yang 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
16S rRNA terbagi menjadi 4 domain
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