etp3.pdf

Eukaryotic Transcription – Part 3 Synthesis of rRNA by RNA polymerase I https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5179497/ SRV 1 RNA Pol I (RNA Pol A) RNA Pol I (Pol I or Pol A) is located in the nucleolus It is responsible for...

Eukaryotic Transcription – Part 3 Synthesis of rRNA by RNA polymerase I https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5179497/ SRV 1 RNA Pol I (RNA Pol A) RNA Pol I (Pol I or Pol A) is located in the nucleolus It is responsible for continuous synthesis of rRNA (18S, 28S, 5.8S) during interphase Pre-rRNA transcription units are arranged in clusters in the genome as long tandem arrays separated by non-transcribed spacer sequences Homologous to RNA pol III and prokaryotic RNA Pol (also RNA pol II, except tail) Crab’s claw structure – all channels, rudder, bridge present Two catalytic Mg2+ The continuous transcription of multiple gene copies of the RNAs is essential for sufficient production of the processed rRNAs, which are packaged into ribosomes Human cells contain 5 clusters of rRNA genes situated on different chromosomes ~300–400 repeats are present in five clusters, located on chromosomes 13 (RNR1), 14 (RNR2), 15 (RNR3), 21 (RNR4) and 22 (RNR5) Each rRNA cluster is known as a nucleolar organizer region, since the nucleolus contains large loops of DNA corresponding to the gene clusters Arrays of rRNA genes loop together to form nucleolus and are known as nucleolar organizer regions SRV 2  Each rRNA gene produces a 45S rRNA transcript called pretranscript or preribosomal RNA or pre-rRNA, which is ~13000 nts long During active rRNA synthesis, the pre-rRNA transcripts are packed along the rRNA genes and may be visualized in electron microscope as ‘Christmas tree structures’ In these structures, the RNA transcripts are densely packed along the DNA and stick out perpendicularly from the DNA The 45S pretranscript is cleaved to give one copy each of 28, 18, 5.8S rRNAs, which are 5000, 2000 and 160 nts long, respectively SRV 3  Pol I is a 14-subunit enzyme that shares five subunits with Pol II and III (Rpb5, Rpb6, Rpb8, Rpb10, and Rpb12) that together with subunits A190, A135, AC40, AC19, and A12.2 form the core Four additional subunits, the heterodimeric A43-A14 stalk, and the A49-A34.5 heterodimer complete the enzyme Transcription by RNA Pol I occurs in nucleolus SRV 4 RNA Pol I promoter These are species specific Since, the numerous rRNA genes in a given eukaryotic cell have essentially identical sequences, its RNA Pol I only recognizes one promoter Yet, in contrast to the case for RNA Pol II and III, RNA Pol I promoters are specific, i.e., an RNA Pol I only recognizes its own promoter and those of closely related species Pol I promoters vary greatly in sequence from one species to another SRV 5 Example, Mammalian RNA Pol I has a bipartite promoter consisting of two transcription control regions (i) Core promoter element It refers to minimal set of sequence elements required for accurate transcription initiation It spans positions -31 to +6 It includes transcription start site and hence overlaps the transcribed region It has a short conserved sequence element, a short AT rich sequence around start point called initiator sequence (Inr) This sequence is essential for transcription (ii) Upstream control element (UCE) or Upstream promoter element (UPE) It is located between residues -187 and -107 bp upstream from the start site The element is GC rich The UCEs are ~85% identical and ~50-80 bp long The sequence is bound by specific transcription factors, which then recruit RNA Pol I to the transcription start site The UCE is responsible for an increase in efficiency of transcription by 10- to 100-fold compared to that from the core element alone SRV 6 SRV 7 Synthesis of precursors of rRNA by RNA Pol I RNA Pol I in nucleolus synthesizes pre-rRNA Pre-rRNA transcription units Arranged in clusters in the genome as long tandem arrays separated by non-transcribed spacer sequences Contain three sequences that encode the 5.8S, 18S and 28S rRNAs Synthesis of rRNA (5.8S, 18S and 28S) involves transcription factors and complexes, for e.g. Upstream binding factor (UBF) and eukaryotic transcription complex called Selectivity factor (SL-1) (similar complex in different species are called TIF-IB, Rib1) SL-1 then helps in recruitment of RNA Pol I on promoter UBF is a specific DNA binding protein, which binds to UCE It greatly stimulates the transcription rate In its absence, a low rate of basal transcription is seen SL-1 contains four subunits: one TBP (TATA binding protein) and three TAFIs (TBP associated factors for RNA Pol I) It helps in recruitment of RNA Pol I on promoter SRV 8 The process of transcription of rRNA (5.8S, 18S and 28S) UBF binding One UBF binds to upstream control element (UCE) of RNA Pol I promoter Other UBF also binds to the upstream region of the core element (core promoter) The sequences in the two UBF binding sites have no obvious similarity One molecule of the UBF is thought to bind to each sequence element UBF-UBF binds by protein-protein interaction causing intervening DNA to form loop between the two binding sites Binding of UBF increases transcription initiation activity by SL-1 (Some are of the view that a single UBF binds to two different sites, viz. UCE and the upstream part of the core element) Selectivity factor binding Selectivity factor (SL-1) binds to and stabilizes the UBF-DNA complex It interacts with the free downstream part of the core element Transcription initiation activity of SL-1 is increased by the binding of UBF RNA Pol I binding SL-1 binding allows RNA Pol I to bind the complex and initiate transcription SRV 9 SRV 10 SRV 11 Nucleolus (plural nucleoli) Most prominent nuclear body Dense, rounded, dark-staining spheroidal bodies Suspended in nucleoplasm Without a limiting membrane Usually eccentric in nucleus Contains chromosomal segments bearing multiple copies of genes specifying rRNA NTS = Non-transcribed spacer ETS = External Transcribed spacer ITS = Internal Transcribed spacer SRV rDNA = rRNA gene (DNA) 12  rRNA genes occupy specific position on chromosome Formed at specific sites called nucleolar organizing regions (NORs) of certain chromosomes, called nucleolar chromosomes Formed around NORs Each NOR contains a cluster of tandemly repeated rRNA genes (rDNA) separated from each other by non-transcribed spacer (NTS) DNA NOR contains blocks of genes that code for rRNA Example in humans 18S, 5.8S, 28S rRNA genes are localized on chromosome number 13, 14, 15, 21, 22 5S rRNA gene is located on chromosome 1 (not present in nucleolus) SRV 13  Three regions IN NUCLEUS Morphologically distinguishable rRNA genes or rDNA Three regions represent sites of progressive stages of rRNA Transcription transcription, processing and ribosome assembly Pre-rRNA or Precursor rRNA Fibrillar center Post-transcriptional Represents chromatin containing rRNA genes or ribosomal processing DNA (rDNA) for coding rRNA Mature rRNAs Dense fibrillar component + Ribosomal proteins (which are imported Represents rRNA from cytoplasm) Granular component Assembly of ribosomal subunits Represents ribosome precursors Transport to cytoplasm through NPC IN CYTOPLASM Ribosome subunits join for translation (i.e., protein synthesis) SRV 14