Molecular Biology

II

Xu Shi Jun, Ph.D

Minimally and Invasive Interventional Department

Henan Cancer Hospital
 Structure of gene-specific
TFs
Common domain:
DNA-binding domain
Transcription-activation domain
 Structure of gene-specific
TFs
Non-common domain ：
Dimerization domain (red arrow)
Ligand-binding domain (blue arrow)

3
 How many gene-specific TFs
are?
 Signaling pathway
12 research fields and 46 pathways
You can find each pathway in CST website

 more than one TF in each signaling pathway

Human genome has ~ 2,600 proteins containing DNA-binding domains

~2,000 TFs are needed for the unique regulation of each gene
Cell Intrinsic Innate Immunity Signaling

Yellow  ellipses represent

transcription factors
MAPK/Erk in Growth and Differentiation

Yellow  ellipses
represent
transcription factors
Regulation of transcription factor activity

Phosphorylation ( 磷酸化 )

Ubiquitylation ( 泛素化 )
Sumoylation (SUMO 修饰 / 类泛素化 )
Methylation ( 甲基化 )
Acetylation ( 乙酰化 )

……
Same Genome,
Differentiated Expression
 Phosphorylation

 Phosphorylase kinase adds phosphate

group
 Phosphatase removes phosphate group

 Change the conformation of TF and form

dimer

 Enter into the nucleus, directly bind to

target DNA

 Increase or repress transcription

 Ubiquitylation
 Signal for protein degradation

 Mediated by ubiquitin (protein),

seven lysine (K) and methionine (M)
residues

 Ubiquitin is connected by K and M

 Enzymes:
E1 (kinase) , E2 (conjugated)
, E3 (ligase)

Deubiquitinating Enzymes
 Sumoylation
 Small ubiquitin-like modifier (SUMO)

 Mediated by SUMO (protein), bind to

the lysine (K) residues of target
protein

 Enzymes: SUMO E1 (kinase) , E2

(conjugated) , E3 (ligase)

 Functions: Regulate the structure of

chromatin, transcription, DNA lesion
repair, et. al.

SUMO proteases
 Methylation
 Targets: DNA, Histone
 Enzymes: transmethylase, demethylase
 Functions: silence gene or protein activity

de novo methylation

Early embryonic development

Core histone of nucleosome: H2A, H2B, H3 and H4

K (lysine) and R(Arginine) residues on N’ of H3,H4

Semiconserved
methylation

DNA replication
 Acetylation
 Targets: proteins, especially histone
 Enzymes: Acetyl transferases, deacetylases
 Functions: regulate protein functioning, structure of chromatin
gene expression

In general, acetylation
promotes gene
transcription.
 Epigenetics
 Regulation of TF is reversible.

 Genome is unchanged, but gene expression is changed.

 Lead to New subject:

Epigenetics
 mRNA splicing
 Along with gene transcription
 Eukaryotic gene is in pieces
 Number and length of introns
are increased during evolution

 Spliceosome mediates splicing

 Final mRNA only has exons.

Splicing signal
 Alternative mRNA splicing
 75% of eukaryotic genes have alternative splicing
 Genes with alternative splicing are increased during evolution
 Generate more than one mRNA coding proteins

One gene, more than one mRNA isoform

 MDK mRNA splicing
The length of first exon and intron varies. Exon skipped

Each of MDK mRNA sequence is called isoform.

 Capping
 Add 5’cap to pri-mRNA
 Materials: 2’-o-methyl nucleotide, GTP, adenosylmethionine

(source of –CH3)

2’-o-methyl nucleotide

GTP
 Polyadenylation

 Add poly (A) to 3’ pri-mRNA, the length of A is 200-500nt.

 Enzyme: poly (A) polymerase

 Material: AMP
 Location: the upstream of transcription terminal site
 Signal: AAUAAA , -20nt of poly(A) site

 Role of cap and poly(A) tail:

Protect mRNAs from degradation, enhance translatability of mRNAs,
transport of mRNAs out of nucleus, efficiency of splicing mRNAs
 Regulation of post-transcription

 ncRNA (non-coding RNA):

 miRNAs (microRNAs)
 Hairpin structure with stem
loop in pri- and pre-miRNA
 20~25nt length, single strand
RNA
 Seed region (6-8nt)
 Completely or partly paired
with target mRNA
 Repression translation or
cleavage mRNA
 Regulation of post-transcription
 ncRNA (non-coding RNA):
 siRNAs (small interfering
RNAs)
 21~25t length, double
strand RNA product
 Completely paired to target
sequence
 Degradation of target mRNA
by cleavage
 Regulation of post-transcription
 ncRNA (non-coding RNA):
 lncRNAs (long non-coding RNAs)
 >200nt length, single strand RNA, similar with mRNA

 Classification:
 Antisense lncRNA
 Intronic transcript
 Large intergenic noncoding RNA
 Promoter-associated lncRNA
 UTR associated lncRNA
 Regulation of post-transcription
 ncRNA (non-coding RNA):
 cicrRNAs (circular RNAs), no cap and poly(A) tail
 More stable than other ncRNA
 Some cicrRNAs serve as sponge of miRNA, release target mRNA
To be continued…