Gene expression in eukaryotes

genitics and molecular biology

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 nucleusin
eukaryotes, nucleoidin bacteria
Translation occurs on ribosomesin 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 exonsand non-coding
sequences called introns
It is the exons that make up the mRNA that gets
translated to a protein

8. Eukaryotic Gene

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 signal12 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
(G
100T
100A
62A
68G
84T
63..) –GU on RNA
has a branch sitenear 3’ end of intron
(one not very conserved consensus
UACUAAC)
ends with an acceptor site consensus.
(12Py..NC
65A
100G
100)
Introns
……AG GUAAGU………A ……(Py) ….. NCAG GU
Donor 5’ splice site Acceptor 3’ splice site

11. The exonsof the transcript region are
composed of:
5’ UTRwith a mean length of 769 bp
AUG (or other start codon)
Remainder of coding region
Stop Codon
3’ UTRwith a mean length of 457 bp
Exons

12. Eukaryotic Promoter

14. Polymerases also use transcription factors
Bind in a specified order, either to promoter or
each other
RNA polymerase IImust 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
TAT AAAA
ATATTTT
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
5
5
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

24. Overall Transcription Process