CHAPTER 3

GENE EXPRESSION IN EUKARYOTES (cont.)

MISS NUR SHALENA SOFIAN

What happen in post-transcriptional event of eukaryotes??

• Eukaryotic genes are mostly interrupted by nonsense DNA - introns

• RNA polymerase is unable to distinguish those genes so it transcribes everything

• Splicing of the nonsense genes occur• In addition, the 5’- and 3’- end of mRNA is tack

with a cap and string of AMPs called poly(A)• These events occur in nucleus before the mRNA

migrates to cytoplasm for translation

“ This is bhgty the human β-globin qhdtchao gene”

• Intervening sequences or introns are unrelated regions on genes coding for mRNA, tRNA and a few for rRNA

• Other parts of the gene, exons, are coding sequences that appear in the final mRNA product

• mRNA may contain 0 -60 introns

Eukaryotic promoter sequences are more variable and often more complex than those of bacteria

For structural genes, at least three features are found in most promoters Transcriptional start site TATA box Regulatory elements

Promoters of Eukaryotic Genes

Usually an adenine

• The core promoter is relatively short– It consists of the TATA box

• Important in determining the precise start point for transcription

• The core promoter by itself produces a low level of transcription– This is termed basal transcription

• Regulatory elements affect the binding of RNA polymerase to the promoter – They are of two types

• Enhancers– Stimulate transcription

• Silencers– Inhibit transcription

– They vary in their locations but are often found in the –50 to –100 region (e.g. GC and CAAT boxes)

Usually an adenine

How do the introns get spliced off?

• RNA splicing – the process of cutting introns out of immature RNAs and stitching together exons to form mature RNAs

• Spliceosome responsible in splicing pre-mRNA in eukaryotes – consists of snRNPs

Mechanisms of splicing introns• U1 binds to 5’ splice site; U2 at branch site• Trimer of three snRNPs: U4/U6 and U5 bind at the

intron region thus forming loop• As the 5’ splice site is cut, its end is attached to

adenosine nucleotide at branch site• 2 exons are bring together; U1 and U4 are released• 3’ splice site is cut; 2 exons are finally connected to

each other• U2, U5, U6 remain attached to the introns (lariat

form)• Intron is degraded; snRNPs are reused

Why need to have introns in genes???

• Alternative splicing – having multiple introns can give variations in splicing, resulting in mRNA that contains alternative combinations of exons

• Occur in different cell types e.g. muscle cells having exons 1-2-4-5-6-7; nerve cells having exons 1-2-3-5-6-7

• Two or more different proteins can derived from the same single gene

Pre-mRNA having 5’-cap and 3’-tail

• Mature mRNA has 5’-cap of 7-methylguanosine

• RNA 5’-triphosphatase removes one of the phosphate groups at 5’-end

• Guanylyltransferase attach GMP from GTP • Methyltrasnferase attaches methyl group to

guanine base

Removes one of the

phosphates

Attaches GMP to the 5’ end

Attaches a methyl group to the

guanine base

Why need capping???

• Require for exiting of mature mRNA from nucleus to cytoplasm

• Cap structure can be recognized by initiation factors that are needed during early stages of translation

• Efficient splicing of introns especially for those that are nearest to 5’-end

3’- poly(A) tail• Needed for:

- mRNA stability- synthesis of polypeptides

• Poly(A) tail is not encoded during transcription; added by polyA polymerase enzyme at 3’-end of the pre-mRNA

• Consensus sequence is AAUAAA downstream from stop codon

• Endonuclease cuts ~20 nt beyond the AAUAAA sequence

• PolymeraseA adds many adenine-containing nucleotides

Appears to be important in the stability of mRNA

and the translation of the polypeptide

Length varies between species

Note: Eucaryotes

Reverse Transcription via reverse transcriptase

• Reverse transcriptase use RNA as template to make cDNA

• The enzyme encodes in retroviruses: RNA to DNA when it integrates into host’s cell

• Otherwise, can use mRNA as template to clone genes

• RNA is mixed with primers of polt-dT in a form of oligonucleotide

• Poly-dT primers complement to the polyA tail of mRNA

• Inclusion of reverse transcriptase and dNTPs to make DNA strand complement to mRNA

• To make other strand of DNA, RnaseH partially degrade the RNA generating short RNA

• The short RNAs are used as primers by DNA polymerase to make second DNA strand

• DNA ligase seals any nicks in the second DNA strans

Why cDNA is useful???• Lacks of introns• Simpler to insert cDNAs directly into vectors to

code a sequence of interest