Struktur Kromosom Prokariotik & Eukariotik PDF

TM-04 Prokaryotic and eukaryotic chromosome structure  Capaian Pembelajaran 1. struktur kromosom prokariotik 2. struktur kromatin 3. struktur kromosom eukariotik 4. kompleksitas genom 5. aliran informasi genetik  PROKARYOTIC CHROMOSOME STRUCTURE The Escherichia coli chromosome ❑ Cont...

TM-04 Prokaryotic and eukaryotic chromosome structure  Capaian Pembelajaran 1. struktur kromosom prokariotik 2. struktur kromatin 3. struktur kromosom eukariotik 4. kompleksitas genom 5. aliran informasi genetik  PROKARYOTIC CHROMOSOME STRUCTURE The Escherichia coli chromosome ❑ Contoh genom prokariotik adalah kromosom E. coli. ❑ Sebagian besar DNA E. coli terdiri dari molekul DNA tungal melingkar tertutup berukuran 4,6 juta pasang basa. ❑ DNA dikemas dalam wilayah sel yang dikenal sebagai nukleoid. ❑ Nukleoid memiliki konsentrasi DNA sangat tinggi, mungkin 30-50 mg/ml, serta mengandung semua protein yang berhubungan dengan DNA, seperti polimerase, represor dan lain-lain. ❑ DNA E. coli terdiri dari 50-100 domain atau loop (Gbr. 1). ❑ Loop berukuran 50-100 kb CP 4.1: menjelaskan struktur kromosom prokariotik  PROKARYOTIC CHROMOSOME STRUCTURE Supercoiling of the genome ❑ Meskipun kromosom E. coli secara keseluruhan adalah superkoil negatif (Lk/Lk° = –0.06), namun beberapa bukti menunjukkan bahwa beberapa supercoiled independently (Fig. 1). CP 4.1: menjelaskan struktur kromosom prokariotik  PROKARYOTIC CHROMOSOME STRUCTURE DNA-binding proteins ❑ Protein-protein terikat membran plasma yang terdapat pada struktur pemersatu domain ada beberapa macam. ❑ Protein yang paling banyak dijumpai adalah HU, suatu protein dimerik (mempunyai dua subunit) yang bersifat basa dan H- NS (dulu disebut H1), suatu protein monomerik netral. ❑ Kedua-duanya mengikat DNA secara nonspesifik dalam arti tidak bergantung kepada sekuens tertentu, dan sering dikatakan sebagai protein mirip histon. ❑ Akibat pengikatan oleh kedua protein tersebut DNA menjadi kompak. ❑ Hal ini sangat penting bagi pengemasan DNA di dalam nukleoid dan stabilisasi superkoiling kromosom. CP 4.1: menjelaskan struktur kromosom prokariotik  CHROMATIN STRUCTURE Chromatin ❑ Kromosom eukariotik masing-masing berisi molekul DNA linear panjang yang harus dikemas ke dalam inti. ❑ Nama kromatin diberikan kepada kompleks DNA-protein yang membentuk kromosom eukariotik. ❑ Struktur kromatin berfungsi untuk mengemas dan mengatur DNA kromosom, dan mampu mengubah tingkat kemasan pada berbagai tahap siklus sel. CP 4.2: menjelaskan struktur kromatin  CHROMATIN STRUCTURE Histones ❑ Komponen protein utama kromatin adalah histon; protein bersifat basa (bermuatan positif) dan berukuran kecil (10-20 kDa) yang mengikat kuat DNA. ❑ Ada empat histon inti, H2A, H2B, H3, H4; dan histon H1 (berukuran sekitar 23 kDa), yang memiliki beberapa sifat dan peran yang berbeda. CP 4.2: menjelaskan struktur kromatin  CHROMATIN STRUCTURE Nucleosomes & The role of H1 ❑ The nukleosom inti adalah unit dasar dari struktur kromosom, yang terdiri dari protein oktamer mengandung masing- masing dengan dua histon inti, dengan 146 bp DNA melilit 1,8 kali histon inti. ❑ Dengan adanya molekul H1, ukuran nukleosom menjadi lebih besar 20 pb (menjadi 166 bp) dan biasanya disebut dengan kromatosom. CP 4.2: menjelaskan struktur kromatin  CHROMATIN STRUCTURE Linker DNA ❑ The linker DNA between the nucleosome cores varies between less than 10 and more than 100 bp, but is normally around 55 bp. ❑ The nucleosomal repeat unit is hence around 200 bp. CP 4.2: menjelaskan struktur kromatin  CHROMATIN STRUCTURE The 30 nm fiber ❑ Chromatin is organized into a larger structure, known as the 30 nm fiber or solenoid, thought to consist of a left- handed helix of nucleosomes with approximately six nucleosomes per helical turn. ❑ Most chromatin exists in this form. CP 4.2: menjelaskan struktur kromatin  CHROMATIN STRUCTURE Higher order structure ❑ On the largest scale, chromosomal DNA is organized into loops of up to 100 kb in the form of the 30 nm fiber, constrained by a protein scaffold, the nuclear matrix. ❑ The overall structure somewhat resembles that of the organizational domains of prokaryotic DNA. CP 4.2: menjelaskan struktur kromatin  EUKARYOTIC CHROMOSOME STRUCTURE The mitotic chromosome ❑ The classic picture of paired sister chromatids at mitosis represents the most highly condensed state of chromatin. ❑ The linear DNA traces a single path from one tip of the chromosome to the other, in successive loops of up to 100 kb of 30 nm fiber anchored to the nuclear matrix in the core. CP 4.3: menjelaskan struktur kromosom eukariotik  LEVELS OF CHROMATIN STRUCTURE CP 4.3: menjelaskan struktur kromosom eukariotik  EUKARYOTIC CHROMOSOME STRUCTURE The centromere ❑ The centromere is the region where the two chromatids are joined and is also the site of attachment, via the kinetochore, to the mitotic spindle, which pulls apart the sister chromatids at anaphase. ❑ Centromeres are characterized by specific short DNA sequences although, in mammalian cells, there may be an involvement of satellite DNA. CP 4.3: menjelaskan struktur kromosom eukariotik  EUKARYOTIC CHROMOSOME STRUCTURE Telomeres ❑ Telomeres are specialized DNA sequences that form the ends of the linear DNA molecules of the eukaryotic chromosomes. ❑ A telomere consists of up to hundreds of copies of a short repeated sequence (5-TTAGGG-3 in humans), which is synthesized by the enzyme telomerase (an example of a ribonucleoprotein) in a mechanism independent of normal DNA replication. ❑ The telomeric DNA forms a special secondary structure, the function of which is to protect the ends of the chromosome proper from degradation. ❑ Independent synthesis of the telomere acts to counteract the gradual shortening of the chromosome resulting from the inability of normal replication to copy the very end of a linear DNA molecule. CP 4.3: menjelaskan struktur kromosom eukariotik CP 4.3: menjelaskan struktur kromosom eukariotik  EUKARYOTIC CHROMOSOME STRUCTURE Interphase chromosomes ❑ In interphase, the genes on the chromosomes are being transcribed and DNA replication takes place (during S- phase). ❑ During this time, which is most of the cell cycle, the chromosomes adopt a much more diffuse structure and cannot be visualized individually. ❑ It is believed, however, that the chromosomal loops are still present, attached to the nuclear matrix. CP 4.3: menjelaskan struktur kromosom eukariotik  EUKARYOTIC CHROMOSOME STRUCTURE Heterochromatin ❑ Heterochromatin comprises a portion of the chromatin in interphase which remains highly compacted, although not so compacted as at metaphase. ❑ It can be visualized under the microscope as dense regions at the periphery of the nucleus, and probably consists of closely packed regions of 30 nm fiber. ❑ It has been shown more recently that heterochromatin is transcriptionally inactive. ❑ It is believed that much of the heterochromatin may consist of the repeated satellite DNA close to the centromeres of the chromosomes, although in some cases entire chromosomes can remain as heterochromatin, for example one of the two X chromosomes in female mammals. CP 4.3: menjelaskan struktur kromosom eukariotik  EUKARYOTIC CHROMOSOME STRUCTURE Euchromatin ❑ The rest of the chromatin, which is not visible as heterochromatin, is known historically by the catch-all name of euchromatin, and is the region where all transcription takes place. ❑ Euchromatin is not homogeneous, however, and is comprised of relatively inactive regions, consisting of chromosomal loops compacted in 30 nm fibers, and regions (perhaps 10% of the whole) where genes are actively being transcribed or are destined to be transcribed in that cell type, where the 30 nm fiber has been dissociated to the ‘beads on a string’ structure. ❑ Parts of these regions may be depleted of nucleosomes altogether, particularly within promoters, to allow the binding of transcription factors and other proteins (Fig. 2). CP 4.3: menjelaskan struktur kromosom eukariotik  EUKARYOTIC CHROMOSOME STRUCTURE DNase I hypersensitivity ❑ Active regions of chromatin, or regions where the 30 nm fiber is interrupted by the binding of a specific protein to the DNA, or by ongoing transcription, are characterized by hypersensitivity to deoxyribonuclease I (DNase I). CP 4.3: menjelaskan struktur kromosom eukariotik  EUKARYOTIC CHROMOSOME STRUCTURE CpG methylation ❑ 5’-CG-3’ (CpG) sequences in mammalian DNA are normally methylated on the cytosine base; however, ‘islands’ of unmethylated CpG occur near the promoters of frequently transcribed genes, and form regions of particularly high DNase I sensitivity. CP 4.3: menjelaskan struktur kromosom eukariotik  EUKARYOTIC CHROMOSOME STRUCTURE Histone variants and modification ❑ The major mechanisms for the condensing and decondensing of chromatin are believed to operate directly through the histone proteins which carry out the packaging. ❑ Short-term changes in chromosome packing during the cell cycle seem to be modulated by chemical modification of the histone proteins. ❑ For example, actively transcribed chromatin is associated with the acetylation of lysine residues in the N-terminal regions of the core histones, whereas the condensation of chromosomes at mitosis is accompanied by the phosphorylation of histone H1. ❑ These changes alter the positive charge on the histone proteins, and may affect the stability of the various chromatin conformations, for example the 30 nm fibers or the interactions between them. ❑ Longer term differences in chromatin condensation are associated with changes due to stages in development and different tissue types. ❑ These changes are associated with the utilization of alternative histone variants, which may also act by altering the stability of chromatin conformations. CP 4.3: menjelaskan struktur kromosom eukariotik GENOME COMPLEXITY Noncoding DNA ❑ This technique allows different classes of repeated DNA to be identified by the effect of multiple copies of a sequence on the rate of renaturation of denatured genomic DNA fragments. ❑ By this method, human DNA can be classified into highly repetitive DNA, moderately repetitive DNA and unique DNA. CP 4.4: menjelaskan kompleksitas genom GENOME COMPLEXITY Reassociation kinetics ❑ This technique allows different classes of repeated DNA to be identified by the effect of multiple copies of a sequence on the rate of renaturation of denatured genomic DNA fragments. ❑ By this method, human DNA can be classified into highly repetitive DNA, moderately repetitive DNA and unique DNA. CP 4.4: menjelaskan kompleksitas genom GENOME COMPLEXITY Unique sequence DNA ❑ This fraction of genomic DNA is the slowest to reassociate on a C0t curve, and corresponds to the coding regions of genes which occur in only one or a few copies per haploid genome, and any unique intervening sequence. ❑ In the E. coli genome, virtually all the DNA has a unique sequence, since it consists predominantly of more or less contiguous (adjacent) single-copy genes. ❑ However, since E. coli has approximately 1000 times less DNA than a human cell, any given sequence has correspondingly more alternative partners at a given concentration, and its reassociation occurs faster, that is at a lower value of C0t. CP 4.4: menjelaskan kompleksitas genom GENOME COMPLEXITY Tandem gene clusters ❑ Moderately repetitive DNA consists of a number of types of repeated sequence. ❑ At the lower end of the repeat scale come genes which occur as clusters of multiple repeats. ❑ These are genes whose products are required in unusually large quantities. ❑ One example is the rRNA-encoding genes (rDNA). ❑ The gene which encodes the 45S precursor of the 18S, 5.8S and 28S rRNAs, for example, is repeated in arrays containing from around 10 to around 10 000 copies depending on the species. ❑ In humans, the 45S gene occurs in arrays on five separate chromosomes, each containing around 40 copies. ❑ In interphase, these regions are spatially located together in the nucleolus, a dense region of the nucleus, which is a factory for rRNA production and modification. ❑ A second example of tandem gene clusters is given by the histone genes, whose products are produced in large quantities during S-phase. ❑ The five histone genes occur together in a cluster, which is directly repeated up to several hundred times in some species. CP 4.4: menjelaskan kompleksitas genom GENOME COMPLEXITY Dispersed repetitive DNA ❑ Much of the moderately repetitive DNA comprises a number of DNA sequences of a few hundred base pairs (SINES, or short interspersed elements) or between one and five thousand base pairs (LINES, or long interspersed elements), each repeated more than 100 000 times and scattered throughout the genome. ❑ The most prominent examples in humans are the Alu and the L1 elements, which may be parasitic DNA sequences, replicating themselves by transposition. CP 4.4: menjelaskan kompleksitas genom GENOME COMPLEXITY Satellite DNA ❑ Satellite DNA, which occurs mostly near the centromeres of chromosomes, and may be involved in attachment of the mitotic spindle, consists of huge numbers of tandem repeats of short (up to 30 bp) sequences. ❑ Hypervariability in satellite DNA is the basis of the DNA fingerprinting technique. ❑ Figure 2 shows an example of a Drosophila satellite DNA sequence, which occurs millions of times in the insect’s genome. CP 4.4: menjelaskan kompleksitas genom GENOME COMPLEXITY Genetic polymorphism ❑ Base changes (mutations) in a gene or a chromosomal locus can create multiple forms (polymorphs) of that locus which is then said to show genetic polymorphism. ❑ The term can describe different alleles of a single copy gene in a single individual as well as the different sequences present in different individuals in a population. ❑ Common types are single nucleotide polymorphisms (SNPs) and simple sequence length polymorphisms (SSLPs). ❑ Where SNPs create or destroy the sequence recognized by a restriction enzyme, restriction fragment length polymorphism (RFLP) will result. CP 4.4: menjelaskan kompleksitas genom  THE FLOW OF GENETIC INFORMATION The central dogma ❑ The central dogma is the original proposal that ‘DNA makes RNA makes protein’, which happen via the processes of transcription and translation respectively. ❑ This is broadly correct, although a number of examples are known which contradict parts of it. ❑ Retroviruses reverse transcribe RNA into DNA, a number of viruses are able to replicate RNA directly into an RNA copy, and a number of organisms can edit a messenger RNA sequence so that the protein coding sequence is not directly specified by DNA sequence. LO 4.5: menjelaskan aliran informasi genetik  THE FLOW OF GENETIC INFORMATION Prokaryotic gene expression ❑ Transcription of a single gene or an operon starts at the promoter, ends at the terminator and produces a monocistronic or polycistronic messenger RNA. ❑ The coding regions of the message are translated by the ribosome from the start codon (close to the ribosome binding site) to the stop codon. ❑ Transfer RNAs deliver the appropriate amino acid, according to the genetic code, to the growing protein chain. LO 4.5: menjelaskan aliran informasi genetik  THE FLOW OF GENETIC INFORMATION Eukaryotic gene expression ❑ In most cases monocistronic messenger RNAs are transcribed from a gene, initiated at a promoter. ❑ The resulting pre-messenger RNA is capped at the 5′-end and has a poly(A) tail added to the 3′-end. ❑ Introns are removed by splicing before the mature mRNA is exported from the nucleus to be translated by ribosomes in the cytoplasm. LO 4.5: menjelaskan aliran informasi genetik LO 4.5: menjelaskan aliran informasi genetik  Next.... DNA replication