Internal Organization of the Nucleus and Ribosome Biosynthesis PDF

Internal Organization of the Nucleus and biosynthesis of ribosomes Asst. Prof. Burcu TÜRKGENÇ Dept. of Medical Biology 1 NUCLEOLUS Prominent Structure within the nucleus 2  NUCLEOLUS (Ribosome production factory) The nucleolus, p...

Internal Organization of the Nucleus and biosynthesis of ribosomes Asst. Prof. Burcu TÜRKGENÇ Dept. of Medical Biology 1 NUCLEOLUS Prominent Structure within the nucleus 2  NUCLEOLUS (Ribosome production factory) The nucleolus, plural: nucleoli) is the largest structure in the nucleus of eukaryotic cells. There is one or more nucleoli 1 µm in diameter Stained with Basic dyes If nuclear chromatin is very condensed It is difficult to observe nucleolus Nucleolus is composed of RNA + proteins (1940) It is the site of ribosomal RNA production 1960 NUCLEOLUS is the site of ▪ rRNA transcription and ▪ Processing Ribosome assembly Cells require large numbers of ribosomes (for protein synthesis) Actively growing cells contain 5-10 million ribosomes  Larger Nucleoli Nucleolus is well developed in cells active in protein synthesis; 1-Young cells (During intense proliferative activity) 2-Cells active in protein synthesis (Acinar cells of pancreas, The pancreatic acinar cell is a highly specialized structure developed for synthesis, storage, and secretion of digestive enzymes) 3-Malignant tumors (rapidly growing)  Nucleolus contains rRNA genes (DNA regions extends into the nucleolus) Pre rRNA transcripts Mature rRNA transcripts Pre ribosomal subunits  Nucleolus is organized around the chromosomal regions Five different human chromosomes; 13, 14, 15, 21, 22 that contain the genes for 5.8S, 18S and 28S rRNAs For the transcription of large numbers of rRNA Cells contain repeated rRNA genes (Multiple copy genes)  5 chromosome pairs which contain rRNA genes , insert into the nucleolus and form NOR regions When the Nucleolus isolated chromosomal foldings remain in the nucleolus Nuclear envelope Human genome contains multiple copies of rRNA genes Ribosomes of higher eukaryotes contain 4 types of rRNAs;5.8S,18S,28S,5S rRNAs The genes for 5.8S, 18S, 28S, rRNAs are clustered in tandem arrays on 5 different human chromosomes Human genome contains about 200 copies of the gene that encodes 5.8S, 18S, 28S, rRNAs Transcribed as a single unit within the nucleolus by RNA polymerase I  Ribosomes Ribosomes are the sites of protein synthesis in both prokaryotic and eukaryotic cells. Both prokaryotic and eukaryotic ribosomes are composed of two distinct subunits, each containing characteristic proteins and rRNAs. E. coli, for example, contain about 20,000 ribosomes, which account for approximately 25% of the dry weight of the cell, and rapidly growing mammalian cells contain about 10 million ribosomes. 10  Ribosomes Ribosomes are also located in mitochondria and chloroplasts of eukaryotic cells; they are always smaller than cytoplasmic ribosomes and are comparable to prokaryotic ribosomes Prokaryotic and eukaryotic ribosomes perform the same functions by the same set of chemical reactions; however, eukaryotic ribosomes are much larger than prokaryotic ones and most of their proteins are different. Mitochondrial and chloroplast ribosomes resemble bacterial ribosomes. 11  large subunit Ribosomes Function protein production small subunit Structure rRNA & protein 2 subunits combine 0.08mm Ribosomes Rough ER Smooth ER  Types of Ribosomes Free ribosomes Found in the cytosol Occur in greater number than bound ribosomes in cells that retain most of their manufactured protein Responsible for proteins that go into solution in the cytoplasm or form important cytoplasmic structural or motile elements 13  Types of Ribosomes Bound ribosomes Found bound to the exterior of the endoplasmic reticulum (ER) constituting rough ER Occur in greater number than free ribosomes in cells that secrete their manufactured proteins (e.g., pancreatic cells, producers of digestive enzymes) Responsible for proteins that become a part of membranes or packaged into vesicles for storage in the cytoplasm or export to the cell exterior 14  Functions of Ribosomes They assemble amino acids to form specific proteins, proteins are essential to carry out cellular activities. The process of production of proteins, the deoxyribonucleic acid produces mRNA by the process of DNA transcription. The genetic message from the mRNA is translated into proteins during DNA translation. The sequences of protein assembly during protein synthesis are specified in the mRNA. 15  Functions of Ribosomes The mRNA is synthesized in the nucleus and is transported to the cytoplasm for further process of protein synthesis. In the cytoplasm, the two subunits of ribosomes are bound around the polymers of mRNA; proteins are then synthesized with the help of transfer RNA. The proteins that are synthesized by the ribosomes present in the cytoplasm are used in the cytoplasm itself. The proteins produced by the bound ribosomes are transported outside the cell. 16  Bacterial Ribosomes The general structures of prokaryotic and eukaryotic ribosomes are similar, although they differ in some details The small subunit (designated 30S) of E. coli ribosomes consists of the 16S rRNA and 21 proteins; the large subunit (50S) is composed of the 23S and 5S rRNAs and 34 proteins. 17  Bacterial Ribosomes (and mitochondrial/chloroplast) The general structures of prokaryotic and eukaryotic ribosomes are similar, although they differ in some details The small subunit (designated 30S) of E. coli ribosomes consists of the 16S rRNA and 21 proteins; the large subunit (50S) is composed of the 23S and 5S rRNAs and 34 proteins. A ribosome is composed of structures called the large and small subunits Each subunit is formed from Note: S or Svedberg units the assembly of Proteins + are not additive rRNA 19  Eukaryotic Ribosomes The subunits of eukaryotic ribosomes are larger and contain more proteins than their prokaryotic counterparts. The small subunit (40S) of eukaryotic ribosomes is composed of the 18S rRNA and approximately 30 proteins; The large subunit (60S) contains the 28s, 5.8s, and 5s rRNAs and about 45 proteins. 20 Human genome contains multiple copies of rRNA genes Ribosome subunits comprise several rRNA molecules, plus a variable number of proteins. Combinations of rRNAs and proteins typically have different sedimentation rates (S), due to condensation. In eukaryotes, 40S Small Subunit includes the 18S rRNA, and a 60S Large Subunit includes the 28S, 5.8S and 5S rRNAs.  Eukaryotic Ribosomes Formed in the cytoplasm during The 40S and 60S subunits are translation assembled in the nucleolus Then exported to the cytoplasm Human genome contains multiple copies of rRNA genes 5S rRNA is not located within the nucleolus There are 2000 copies of the gene that encodes 5S rRNA 5S rRNA genes are present in a single cluster on chromosome 1. Transcription of the 5S rRNA takes place out of the nucleolus (catalyzed by RNA polymerase III )  Transcription and Processing of rRNAs 1-Transcription of Large 45S pre-rRNA (which contains 18S, 5.8S, 28S rRNAs) 2-Processing Cleavages 45S rRNA splits to form separate precursors of the large and small ribosomal subunits the 18S (small subunit) 5.8S and 28S rRNAs (large subunit) Chemical Modifications 3-Association of ribosomal proteins with rRNA (Ribosome assembly) Begins while the pre-rRNA is still being synthesized The chemical modification and nucleolytic processing of a eucaryotic 45S precursor rRNA molecule into three separate ribosomal RNAs.  The processing of pre rRNA requires both proteins and RNAs Nucleolus contains 300 proteins and About 200 small nucleolar RNAs (snoRNAs) that function in preRNA processing  Other functions of NUCLEOLUS Nucleolus is also the site where other RNAs are produced and other RNA-protein complexes are assembled For example U6 SnRNP (small nuclearribonucleoprotein) have function in pre mRNA Splicing CHROMATIN (DNA+histone and nonhistone proteins) Chromatin is a packaging material for DNA. If not packed well, it can tangle around itself or get damaged during cell division. Stained with Basic dyes ( + ) Feulgen reaction ( Specific stain for the identification of DNA) 1-Euchromatin 2-Heterochromatin Chromatin 1-Euchromatin (Lightly stained) Invisible with L.M Visible with EM less condensed, uncoiled, dispersed form of chromatin Active form Transcriptionally active DNA surface is available for transcription *Euchromatin contains most of the potentially active (inducible) genes of a cell.  2-Heterochromatin Dark staining, visible form of chromatin Localizes to the nuclear periphery Some of the chromatin fibers; coiled, condensed and folded into dense, tightly packed masses Inactive form of chromatin DNA packed into heterochromatin is inactive in transcription In a typical mammalian cell, more than 10 % of the genome is packaged into heterochromatin  2-Heterochromatin it is also found in specific chromosomal regions; the centromeres and telomeres Figure: Condensed metaphase chromosome Fine structure of Chromatin  Fine structure of Chromatin Complexes of DNA and Protein are called CHROMATIN Major proteins of chromatin are the histones DNA + Protein (Histone and non histone proteins) DNA of eukaryotic cells is bound to small basic proteins; histones  The total extended length of DNA in a human cell is 2 meters This DNA must fit into a nucleus with a diameter of only 5 to 10 µm Histones package the DNA in an orderly manner in the cell nucleus  Fine structure of Chromatin The basic structural unit of chromatin (nucleosome) was first described by Roger KORNBERG in 1974 Chromatin is isolated from nucleus and spread on lower concentration of salt solution Examined in EM ; 10 nm Chromatin fibers have a beaded appeaerance 30 nm (A) and 10 nm (B) chromatin fibers Electron microscopical appearience  Fine structure of Chromatin 1- 10nm chromatin fiber Bead like structures; nucleosomes ( core histones+DNA) Nucleosome is a disc shaped structure which consists of DNA coiled around an octamer (8 histone molecules) of core histones There are 5 major types of histones Histones; 1-H2A, H2B, H3, H4 (nucleosomal histones) 2-H1 histones (linker histone)  10 nm chromatin fiber First level of folding involves coiling of DNA around a protein core to form a bead-like structure called nucleosome Shortens DNA length 6 fold relative to naked DNA Chromatin is composed of repeating units (Nucleosomes) Packaging of DNA with histones yields a chromatin fiber 10 nm in diameter Beads spaced at intervals of 200 base pairs 10nm chromatin fiber Nucleosome core particle consists of A Histone octamer consisting of two copies of each of (H2A,H2B,H3,H4) ( 8 histone molecules) DNA winds around octamer (~ 2 turns 146bp) Between the 2 Nucleosomes → linker DNA (~80bp). (H1 binds to linker DNA between the nucleosomes close to nucleosomal core  two copies each of H2A, H2B, H3, H4 - DNA wraps 146 bp (1.75 turns) around the core H1 attaches to the linker region and changes the conformation; required for chromatin formation Histone Octamer Each chromosome contains a single molecule of DNA This DNA is wound around histones These proteins have lots of lysine and arginine residues, making them very positively charged at pH 7  30 nm Chromatin fiber Chromatin can be further condensed by coiling into 30 nm fibers 30 nm Chromatin fiber (second level of DNA packaging) 10 nm chromatin fiber is folded into a thicker filament 30 nm Chromatin fiber Shorten DNA length 50 fold relative to naked DNA Interactions between H1 histones play important role in this stage of chromatin condensation Structure of 30nm fiber remained unknown until 2005 30 nm Chromatin fibers; String of nucleosomes is folded into a thicker filament There are approximately 6 nucleosomes in each turn of the helix (Solenoid) Chromatin is organized as 30nm chromatin fiber at interphase Zigzag model of the 30-nm chromatin fiber 46