* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
Download nuclear structure (2): the nucleolus
DNA supercoil wikipedia , lookup
Cre-Lox recombination wikipedia , lookup
Gene expression profiling wikipedia , lookup
DNA polymerase wikipedia , lookup
Transcription factor wikipedia , lookup
Nucleic acid double helix wikipedia , lookup
Epigenomics wikipedia , lookup
Metagenomics wikipedia , lookup
Point mutation wikipedia , lookup
Designer baby wikipedia , lookup
Human genome wikipedia , lookup
X-inactivation wikipedia , lookup
Extrachromosomal DNA wikipedia , lookup
History of genetic engineering wikipedia , lookup
Microevolution wikipedia , lookup
Long non-coding RNA wikipedia , lookup
Polycomb Group Proteins and Cancer wikipedia , lookup
Non-coding DNA wikipedia , lookup
RNA interference wikipedia , lookup
Artificial gene synthesis wikipedia , lookup
Messenger RNA wikipedia , lookup
Vectors in gene therapy wikipedia , lookup
Short interspersed nuclear elements (SINEs) wikipedia , lookup
Nucleic acid analogue wikipedia , lookup
Therapeutic gene modulation wikipedia , lookup
Polyadenylation wikipedia , lookup
Epigenetics of human development wikipedia , lookup
Nucleic acid tertiary structure wikipedia , lookup
Deoxyribozyme wikipedia , lookup
RNA silencing wikipedia , lookup
RNA-binding protein wikipedia , lookup
History of RNA biology wikipedia , lookup
Epitranscriptome wikipedia , lookup
1 NUCLEAR STRUCTURE (2): THE NUCLEOLUS Reading: pp. 538-547; p. 95 (Svedberg units) More than 95% of the RNA in a eukaryotic cell is rhe RNA that forms part of the structure of ribosomes. This is the ribosomal RNA (rRNA). The “size” of macromolecules such as proteins and RNA can be determined by how rapidly the molecule moves to the bottom of the centrifuge tube during ultracentrifugation. Actually the “size” determined by this method is a reflection of both the mass and the shape of the molecule. The units used are Svedberg units (S). The larger the S value, the larger the molecule. But S values do not add up in a linear fashion because of the influence of both mass and shape on the detrmination of the S value. The important facts in the previous diagram are: (1) A functional ribosome consists of a large subunit and a small subunit. (2) Together, the two subunits possess about 80 different ribosomal proteins. (3) The large subunit contains three ribosomal RNA molecules. The sizes of the ribosomal RNAs are 5S, 5.8S, and 28S. (4) The small subunit contains one ribosomal RNA that is 18S in size. The ribosomal RNA (rRNA) has two functions; (1) It serves as the skeleton for the attachment of the ribosomal proteins. (2) It is actually the rRNA that catalyzes the formation of the peptide bonds. The ribosomal proteins give the required shapes to the ribosomal subunits to allow the proper binding of the transfer RNA molecules, mRNA, and exit of the growing polypeptide. 2 The ribosomal subunits are synthesized in nucleoli. Some of the events related to the synthesis of the ribosomal subunits that occur in the nucleolus are: (1) Transcription of the ribosomal DNA genes (rDNA genes) that occur in the nucleolus.The transcription is catalyzed by RNA polynerase I (RNA pol I). (2) The RNA molecule that results from this transcription has a size of 45S. It is cut to form three of the four ribosomal RNA (rRNA) molecules that are rquired for the ribosomal subunits. (3) Proteins are added to the nascent RNA transcript. These proteins include: ribosomal proteins that will remain associated with the rRNA; proteins (enzymes) that cut the RNA transcript to form the 5.8S, 18S, and 28S ribosomal RNA molecules; and proteins that help the assembly of the ribosomal subunits (such as nucleolin). (4) The 5S ribosomal RNA is transcribed from a gene located outside the nucleolus. This ribosomal RNA must enter the nucleolus so it can be assembled into the large subunit. The processing of the 45S ribosomal RNA transcript. There are about 400 copies of the gene for the 45S rRNA transcript in each human somatic cell. The reasons for the large number of these genes are: (1) So many ribosomes must be made. (2) The only possible amplification step in the making of RNA is the possibilty of more than RNA polymerase molecule transcribing the gene at a given time. (For proteins there is the second amplification possibilty of having multiple ribosomes on a messneger RNA (mRNA) molecule.) 3 Synthesis of rRNA in the nucleolus COMPLETED 45S rRNA TRANSCRIPT DIRECTION OF TRANSCRIPTION PROMOTER SEQUENCE 5.8S, 18S AND 28S rRNA CUTTING RNA POLYMERASE 1 TERMINATION SEQUENCE ON THE TEMPLATE DNA STRAND TRANSCRIPTION UNIT FOR THE 45S rRNA TRANSCRIPT In this case the amplication steps are: (1) Multiple RNA polymerase molecules on the transcription unit (the 45S rRNA gene), transcribing simultaneously. (2) Multiple copies (400 per human somatic cell) of the 45S rRNA gene. In amphibians, which may large eggs with a lot of cytoplasm containing a lot of ribosomes, extrachromosomal nucleoli can be formed. To from these the nucleolar organizing regions (NORs) (chromosomal regions containg the 45S rRNA genes) are replicated (DNA replication) a hundred times or more. These NORs become free of the chromosomes and each one then forms a small extrachromosomal nucleolus. This is a light microscope view of anuclues isolated from an amphibian oocyte (Xenopus, the clawed toad). The hundreds of extrachromosomal nucleoli can ce seen. Formation of such large numbers of nucleoli is clearly another from of “amplificatio” in terms of making ribosomes more rapid;y. 4 Synthesis of most proteins DIRECTION OF TRANSCRIPTION pre-mRNA TRANSCRIPT PROMOTER SEQUENCE mRNA SPLICING OUT OF INTRONS RNA POLYMERASE 1 RNA POLYMERASE II GENE DIRECTION OF TRANSLATION PROTEIN mRNA MULTIPLE RIBOSMES ON ONE mRNA MOLECULE (CALLED A POLYSOME) 5 DIRECTION OF TRANSCRIPTION The top panel shows a nucleolar spread as seen in the electron microscope. Note how many “christmas trees” there are. Each “tree” is a 45S RNA gene being rapidly transcibed. Ans this is only a small sample of the entire nucleolar spread! Notice that there a a non-transcribed region between each “tree” (each 45S RNA gene). The lower panel shows a higher magnification of two of the 45S rRNA genes. Note the following; (1) The promoter is located at the upper left hand end of the genes. (2) The black “dots” on the DNA (at the bottom of each “branch”) are the RNA polymerase molecules. (3) The “branches” are the nascent 45S rRNA molecules. (4) At various locations along each “branch” (each nascent 45S rRNA molecule) are black dots. These are places where proteins have bound. (5) The nascent RNA molecules do not appear to be as long as the DNA template on which they transcribed. Of course they have to be the same length before any processing occurs. The reason they do not appear to be the same length is that the proteins that have already been added cause the RNA transcript to “bunch up”. (6) The black dots in the non-transcibed spacer region are not RNA polymerase molecules. Instead they are nucleosomes (the two tures of DNA double hexix wrapped around 8 histone molecules. 6 DIRECTION OF TRANSCRIPTION The “trees” are diagrammatic, not actual genes being transcribed as are shown in the previous figure. Note the following; (1) There are two types of “spacer” in in the DNA template. One spacer is a length of DNA that is not transcribed and which separtes the individual copies of the 45S rRNA gene. Not surprisingly, these are called the non-transcribed spacers. Then there are the spacers (yellow bars) that separate the DNA sequences that actually have the information for the synthesis of the 5.8S, 18S, and 28S rRNA molecules. These are spacers are actually transcribed to form the 45S RNA transcript. These transcribed spacers must then be cut out of the 45S RNA trnascript. (2) Each gene has a promoter and a small sequence at the downstrean end that signals “termination” for the RNA polymerase molecules. 18S 5.8S 28S NON-TRANSCRIBED SPACER NON-TRANSCRIBED SPACER THE 45S DNA TRANSCRIPTION UNIT (TEMPLATE) TRANSCRIPTION CUTTING OUT OF THE TRANSCRIBED SPACERS 18S rRNA 5.8S rRNA 28S rRNA 7 The nucleolar organizing region (NOR) In the above review diagram of the role of the nucleolus in the synthesis of the ribosomal subunits you can see the term “loop of nucleolar organizer DNA”. This is really just another term for “all the 45S rRNA genes and the non-transcibed spacer DNA”. This is usually called the nucleolar organizing region (NOR). A nucleolus can form at each of these regions, and in human somatic cells just after their division this is the case. In fact, 10 nucleoli can be observed in these cells just after division. In humans, the 10 NORs are located on 5 separate homologous pairs of chromosomes (for a total of 10 seoarte chromosomes!). later in the life cycle of the cells these individual nucleoli usually coalesce to from large nucleolus (see the diagram below). 8 This diagram shows the appearance of the nucleolus (nucleoli) during the life cycle of somatic cell capable of cell division. 9 Key words (inluding for previous set of notes) transcription factory replication factory painting chromosomes nuclear lamina intermediate filaments nuclear envelope inner nuclear membrane outer nuclear membrane heterochromatin euchromatin chromatin telomer fluorescence in situ hybridization (FISH) biotin biotinylation of a deoxyribonucleotide avidin fluorophore covalently bound to avidin separation of DNA strands (“melting”) RNA polymerase DNA polymerase nucleolus nucleolar organizing region 45S ribosomal RNA genes 45S ribosomal RNA transcript transcribed spacer non-transcribed spacer processing (cutting) of 45S RNA transcript about 80 ribosomal proteins 45S rRNA 5.8S rRNA + 18S rRNA + 28S rRNA 5S ribosomal RNA genes (extranucleolar) fibrous region of nucleolus (rDNA + 45S rRNA transcript) granular region of nucleolus (developing large and small ribosomal subunits0 extrachromosomal nucleoli of amphibians “amplification steps” for RNA and protein synthesis polysome ( = polyribosome) small ribosomal subunit (40S) large ribosomal subunit (60S) Svedberg unit (by ultracentrifugation) ribonulceoprotein particle (specific proteins bound to specfic RNA molecules) nucleosome histone