Molecular Biology

III

Xu Shi Jun, Ph.D

Minimally and Invasive Interventional Department

Henan Cancer Hospital
 Translation
From mRNA to protein (the second genetic code)
It costs almost 80% energy and 50% weight of each cell.

Direction: 5’ to 3’
Core elements: mRNA (template, Coding information)
tRNAs (transfer RNAs, Decoding & Transferring information)
rRNA (ribosome RNAs, Protein Synthesis Machine) and ribosome

Process: initiation, elongation and termination.

https://www.bilibili.com/video/av498647359/
 Codon
 Definition: three adjacent nucleotides (5’ to 3’) on mRNA become one
amino acid.
Amino acid is the basic unit of protein.

The arrangement of codons

determine the sequence of
amino acids.

First codon: AUG (Met, methionine)

Stop codon: UAA, UAG, UGA

Totally, 20 kinds of amino acids form

all proteins.
3
 tRNA
 Key element in translation
 Adapter: transfer ATCGs to polypeptide
 Enzyme: aminoacyl-tRNA synthetase

Secondary structure Tertiary structure

Amino acid
 Ribosome
 Ribosome: big (80S) and small subunit (40S), consist of rRNA and proteins
 Small subunit: include 16S rRNA, decode center
codon binds to anticodon located on tRNA
 Big subunit: include 5S, 28S and 5.8S rRNA, peptidyl transferase
activity center, ligand each amino acid to form polypeptide
 SD sequence: small ribosomal subunit binding site,
3-9nt upstream of AUG

 16S rRNA recognizes SD sequence

*S: sedimentation coefficient, the criteria of RNA

molecular weight.
 Ribosome
Big ribosomal subunit:
 P site: peptidyl-tRNA binding, the first tRNA binding site
 A site: aminoacyl-tRNA binding, the second tRNA binding site
 E site: uncharged tRNA exiting, the third tRNA binding site

 Translation order:
P-A-E
E P A

 Final product:
polypeptide, export to cytoplasm and modified by endoplasmic reticulum
and Golgi complex to become functional proteins.
 The speed of life

 Speed for DNA replication: 200-1,000nt per second

PCR primer design
 Speed for DNA transcription: 50-100nt per second
qRT-PCR primer
 Speed for mRNA translation: 2-4nt amino acid per second

 The faster process, the less energy and proteins are needed.
 Post-translational regulation

 Cut off the methionine (first amino acid) on N’ of polypeptide

 Forming disulfide bonds to fold polypeptide, then protein

 Modification: phosphorylation, glycosylation, methylation,

ethylation, hydroxylation, et. al.

 Cut off intein (non-functional fragment of polypeptide)

Post-translational regulation

miRNA

Annu. Rev. Biochem. 2010. 79:351–79

 Classically experimental
Methods
I. Detection

 DNA : PCR (amplification of certain DNA sequence) with primers

southern blot (genomic DNA specific sequence mapping) with
marked probes, 32P or biotin.

Agarose gel electrophoresis to separate different size of DNA (kb)

Southern blot image

PCR product image Mieke Dewerchin, J Clin. Invest.,1996
 Classically experimental
Methods
I. Detection
 RNA : quantitive real-time PCR (qRT-PCR) with fluorophore-primers
northern blot (specific genes at specific stages of growth and
development or under stress or pathological environment)
with
Northern blot image
marked probes, 32P or biotin.
qPCR data analysis Northern blot result

Hui Xu, Shi-Jun Xu, eLife, 2019 Hui Xu, Hepatology, 2010
Tao Yang, BMC Genomics, 2022
 Classically experimental
Methods
I. Detection
 Protein : western blot (WB), ELISA (for secretory proteins), second antibody
marked with horse radish peroxidase (HRP)
Immunofluorescence (IF), second antibody with fluorophore
Immunohistochemistry (IHC), second antibody with HPR, color
developing agent IF
WB
IHC

Hong-Tao Hu, CANCER RES TREAT

Hui Xu, Shi-Jun Xu, eLife, 2019 , 2020

Hui Xu, Jie-Hua He, Shi-Jun Xu, J Cell Biochem, 2018

 Classically experimental
Methods
II. Interaction
 Protein with protein: protein A affect protein B expression
immunoprecipitation (IP):
Goal: confirming protein A can directly bind to protein B.
Method: magnetic bead-antibody A-cell lysis, detect protein B by WB.
co-immunoprecipitation (co-IP):
Goal: confirming protein A directly bind to protein C then recruit protein B.
Method: magnetic bead-antibody A-cell lysis, detect protein C and B by WB.

IP result: Sp1 directly binds to α-eEF1A1

Chunxian Zeng, RNA Biol., 2020

 Classically experimental
Methods
II. Interaction

 Protein with DNA: Transcriptional factor A affect gene B transcription

Dual-luciferase reporter assay:
Goal: confirming protein A bind to the promoter of gene B then change its
transcription.
Method: overexpression or downregulation of protein A, luciferase reporter
vector with DNA sequence of protein B promoter,
dual-luciferase assay system for detection.

Hui Xu, Shi-Jun Xu,

eLife, 2019
 Classically experimental
Methods
II. Interaction
 Protein with DNA: Transcriptional factor A affect gene B transcription
chromatin-immunoprecipitation (ChIP):
Goal: finding the directly target DNA of transcription factor A in vivo.
Method: bead-antibody A-cell lysis, DNase digestion, protease digestion,
purification of DNA, qRT-PCR and agarose gel detection or sequencing.

Hui Xu, Shi-Jun Xu, eLife, 2019

 Classically experimental
Methods
II. Interaction

 RNA with protein: RNA binds to RNA binding protein (RBP)

RNA immunoprecipitation(RIP)
Goal: finding the RBPs of certain RNA or the RNAs of certain RBPs.
Method: marked RNA, bead-RNA-cell lysis, digestion RNA, WB for detection.
RBP antibody, bead-RBP-cell lysis, digestion RBP, qRT-PCR for
detection.
 Classically experimental
Methods
II. Interaction

 miRNA with protein: whether gene A is the target of miRNA

Dual-luciferase reporter assay:
Goal: confirming miRNA directly bind to protein A then change its expression.
Method: overexpression or downregulation of miRNA, luciferase reporter
vector with DNA sequence of core functional region of protein B,
dual-luciferase assay system for detection.

Hui Xu, Shi-Jun Xu, eLife, 2019 Nat. Rev. Genet. 5(7):522-31,2004.
End