Gene expression in eukaryotes
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1) Gene expression in eukaryotes involves transcription occurring in the nucleus, with mRNA then being transported to the cytoplasm for translation by ribosomes. 2) Eukaryotic genes contain both coding exons and non-coding introns, with the exons needing to be spliced together to form mRNA before translation. 3) Eukaryotic transcription initiation requires multiple transcription factors binding in a specific order to the promoter region upstream of a gene, with RNA polymerase II then initiating transcription.
Gene expression in eukaryotes
- 1. Gene Expression in Eukaryotes M.Prasad Naidu MSc Medical Biochemistry, Ph.D.Research Scholar
- 2. Outline Central dogma in Eukaryotes Nature of Genes in Eukaryotes Initiation and Elongation of Transcription RNA Processing
- 3. Eukaryotic Transcription Transcription occurs in the nucleus in eukaryotes, nucleoid in bacteria Translation occurs on ribosomes in the cytoplasm mRNA is transported out of nucleus through the nuclear pores
- 4. In Eukaryotes (cells where the DNA is sequestered in a separate nucleus) the exons must be spliced (many eukaryotes genes contain no introns! Particularly true in ´lower´ organisms). mRNA (messenger RNA) contains the assembled copy of the gene. The mRNA acts as a messenger to carry the information stored in the DNA in the nucleus to the cytoplasm where the ribosomes can make it into protein. Eukaryotic Central Dogma
- 5. ~6 to 12% of human DNA encodes proteins (higher fraction in nematode) ~90% of human DNA is non-coding ~10% of human DNA codes for UTR Eukaryotic Genome - Facts
- 6. Untranslated regions (UTRs) • Introns (can be genes within introns of another gene!) •Intergenic regions: - Repetitive elements - Pseudogenes: Dead genes that may (or may not) have been retroposed back in the genome as a single-exon “gene” Non-Coding Eukaryotic DNA
- 7. Coding and Non-coding Sequences In bacteria, the RNA made is translated to a protein In eukaryotic cells, the primary transcript is made of coding sequences called exons and non-coding sequences called introns It is the exons that make up the mRNA that gets translated to a protein
- 9. Eukaryotic Nuclear Genes Genes transcribed by RNA Pol II •Upstream Enhancer elements. •Upstream Promoter elements. •GC box (-90 nt) (20 bp), CAAT box (-75 nt) (22 bp) •TATA promoter (-30 nt - 70%, 15 nt consensus (Bucher et al., 1990) •Transcription initiation. •Transcript region, interrupted by introns. Translation Initiation (Kozak signal 12 bp consensus: 6 bp prior to initiation codon) •polyA signal (AATAAA, 99%)
- 10. •Transcript region is interrupted by introns Each intron (on DNA): starts with a donor site consensus (G100T100A62A68G84T63..) – GU on RNA has a branch site near 3’ end of intron (one not very conserved consensus UACUAAC) ends with an acceptor site consensus. (12Py..NC65A100G100) Introns ……AG GUAAGU ………A ……(Py) …..NCAG GU Donor 5’ splice site Acceptor 3’ splice site
- 11. The exons of the transcript region are composed of: 5’ UTR with a mean length of 769 bp AUG (or other start codon) Remainder of coding region Stop Codon 3’ UTR with a mean length of 457 bp Exons
- 14. Polymerases also use transcription factors Bind in a specified order, either to promoter or each other RNA polymerase II must be phosphorylated before it can start synthesizing RNA Sequences of Eukaryotic promoter
- 15. RNA polymerase I- makes precursors for ribosomal RNAs (except for smallest subunit) RNA polymerase II- mRNA and snRNAs (involved in RNA processing) RNA polymerase III- variety of RNAs: smallest rRNA subunit, tRNA precursors Each uses a different promoter (DNA sequences that direct polymerase to begin tran- scribing there) Promoters are “upstream” from coding sequence Eukaryotic RNA polymerases
- 16. Initiation in Eukaryotes TRANSCRIPTION RNA PROCESSING TRANSLATION DNA Pre-mRNA mRNA Ribosome Polypeptide Eukaryotic promoters T A T A AA A A T A T T T T TATA box Start point Template DNA strand 5 3 3 5 Several transcription factors Additional transcription factors Transcription factors 5 3 3 5 1 2 3 Promoter 5 3 3 55 RNA polymerase II Transcription factors RNA transcript Transcription initiation complex Several transcription factors must bind to promoter sequences upstream of the gene Then RNA polymerase can bind TATA box
- 17. Requirements for initiation of Transcription
- 18. Transcription Factors – Order of their binding
- 20. General transcription factors Eukaryotic Transcription initiation TATA binding protein (TBP)/TFIID binds to TATA box (-25)
- 21. Role of Enhancers in Initiation