CH1131 Biomolecular

Engineering
Week 2 (Aug 17 & 19)
Making of Parts I - Transcription
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Learning Outcome
DNA / RNA
Process of Transcription
Process of Translation
Gene and Function

Outline of Lectures on DNA

DNA and Gene (1)

DNA Replication (3)

Gene, Transcription and Function (2)

From Gene to Protein: information flow

Introduction
(Genomics)

(Transcriptomics)

DNA

Protein

RNA
Transcription

(Proteomics)

Translation

Transcription: A process in which DNA provides the

information to produce messenger RNA

Translation: Protein synthesis from messenger RNA

Control of Gene expression

Ribonucleic Acids: definition

Nucleotide (DNA): deoxyribose

sugar + nitrogeneous base +
phosphate
Nucleotide (RNA): ribose sugar +
nitrogeneous base + phosphate

Ribonucleic Acids:
structural perspective

Nucleic acid

Nitrogeneous base: 1.
Pyrimidines: thymine (T) and
cytosine (C), 2. Purines:
guanine (G) and adenine (A)

Complementary bases: A = T
(2 H bonds), G = C (3 H
bonds), purines with
pyrimidines.
Joined by H bonds, forming
sugar-phosphate backbones.

Chemical polarity of DNA

Nucleotides are asymmetrical.
Linked to DNA chain in a head-to-tail fashion.
Hence entire DNA strand has a chemical
polarity.

Fine Difference between DNA

and RNA

RNA

3 Primary Functions of DNA

1. Storage of genetic information:
Determines all inheritable characteristics.
Nucleotide sequences within a Coding
Region determines protein to synthesized.
2. Replication and inheritance:
DNA must be copied accurately via replication
Transmit genetic information to daughter cells
3. Expression of genetic message:
Genes encode proteins consisting of amino
acids.
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Genes

Definition: Entire nucleic

acid sequence necessary
for the synthesis of a
functional polypeptide

Hereditary
Information
encoded in
DNA

http://www.youtube.com/watch?
v=zwibgNGe4aY What is DNA
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RNA Can Form Secondary Structures Based

on Sequence Complementarities

Even though RNA is singlestranded, they often fold

back on themselves (based
on complementarity of
nitrogeneous bases) to
produce molecules of
double-stranded segments

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Classes of RNA
3 major classes of RNA: rRNA, tRNA, mRNA
Ribosomal RNA (rRNA)

75% - 80% of cellular RNA.

rRNA, together with proteins, form
ribosomes.
Produced in nucleus.

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Transfer RNA (tRNA)

10% - 15% of cellular RNA.

Decodes mRNA during translation (protein
synthesis).

Messenger RNA (mRNA)

5% - 10% of cellular RNA.

Carries the genetic information for protein
synthesis.
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General Features of
Transcription
4 Typical features of Eukaryotic Transcription:

RNA Polymerase
Initiation: Promoter
Elongation
Termination

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RNA Polymerase (RNAP)

RNA Polymerase (DNA-dependent RNA

Polymerase) catalyzes transcription in all cells

Function is to incorporate nucleotides to RNA

strands during transcription

The RNA formed is complementary to one of the

DNA strands (the template).

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Transcription in Eukaryotes
mRNA synthesized by RNA polymerase II
Associated with proteins called general transcription factors
(GTFs).

First step: Pre-initiation complex assembly

Specific binding of a protein to the eukaryotic promoter at 24 to 32
bases upstream of the initiation site known as the TATA box.
The binding protein is called TATA-binding protein (TBP).
It is a subunit of transcription factor for polymerase II, fraction D
(TFIID).

TATA box

Initiator

http://www.youtube.com/watch?v=bk7PW1FKMTI Transcription

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Transcription in Eukaryotes:
initiation

https://www.google.com.sg

http://www.youtube.com/watch?v=P6Nyce-4oG4 Eukaryote Transcription

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Transcription in Eukaryotes: elongation

Second step: Elongation (Transcription begins!)

https://www.google.com.sg

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Transcription in Eukaryotes: termination

Termination includes cleavage of messenger RNA and
addition of poly-adenosine tail (more than 50
adenosines)

elongation

termination

http://en.wikipedia.org/wiki/File:Simple_transcription_termination1.svg

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Transcription in Eukaryotes: modification

1. Contains non-coding segments called untranslated regions
(UTRs), at ends 5 and 3 which play regulatory roles
2. Consists of methylated guanosine cap at 5 end and a string
of adenosine residues of poly(A) tail at 3 end.

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Transcription in Eukaryotes: modification

5 caps: A dual active enzyme (triphosphatase and
guanylyltransferase) binds to 5 end.
Methyl group addtion by methyltransferase to terminal
guanosine cap and ribose of terminal nucleotide.
A protein complex called CBC binds to complete capping
Poly(A) tails: At 3 end, endonuclease cleaves pre-RNA 20
nucleotides downstream from recognition site from
sequence AAUAAA.
Poly(A) polymerase adds adenosine residue.

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Transcription in Eukaryotes: modification

Functions of 5 end cap
Prevents the 5 of mRNA being digested by
endonucleases.
Aids in mRNA transport out of the nucleus.
Plays a role in mRNA translation.
Functions of 3 end poly(A) tail
Protects mRNA from premature degradation by
exonucleases.
http://www.youtube.com/watch?v=vi-zWoobt_Q
Regulated Transcription

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Summary
DNA (RNA) structures
Gene structure and organization
Transcription (RNA polymerase, promoter,
elongation, termination)

Co-transcriptional Modifications
(Methylguanosination at 5 end and polymerization
of adenosine at 3 end)

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Tutorial Topics
Propose a simple experiment to test the
Hypothesis: gene determines function

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Making of Parts II - Translation

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Learning Objective

Transcription

Translation

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Learning Outcome

tRNA and anticodon

Open Reading Frame in DNA
Process of Translation
Translation of Membrane Proteins
Protein Folding Mechanism

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Genetic code and tRNA

DNA sequence carries information for protein
It is represented as Genetic Code
Each amino acid (AA) is encoded by 3 sequential
nucleotides: codon for AAs were nucleotide triplets
Decoding information in mRNA is done by tRNAs (tRNAs
are adapters: specify AA and consists of anticodon)

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tRNAs
Transcribed by RNA polymerase III.
Genes in DNA (tDNA) encoding
tRNAs
organized in tandem array.
separated by non-transcribed spacer
sequences.
found in small clusters scattered
around genome.

Matured tRNAs have triplet

nucleotides CCA at 3 end.
tRNAs play key role in translation
(protein synthesis).

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tRNAs structure
1. Parts of molecule have
sequences complementary
to that at other parts:
cloverleaf shape (2D), 2
double strands in L-shape
2. AA always carried by
adenosine (A) at 3 end
3. Anticodon at position 3 to 5 of
invariably composed loop
of 7 nucleotides.

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The Genetic code

4 bases for triple nucleotides 43= 64 possible mRNA codons combinations.

In fact 61 different codons specify 20 AAs (Wobble hypothesis), 3 stop
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codons.

Wobble hypothesis
Wobble hypothesis: the steric requirement between
anticodon (in tRNA) and codon in (mRNA) is very strict for
the first 2 positions and more flexible for 3rd position.

3end
5end
Anticodon
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Wobble hypothesis
2 codons specifying same AA but differ at 3rd position
should use same tRNA in protein synthesis.
U of anticodon (5 end) can pair with A or G of mRNA
codon (3 end), while G can pair with U and C, I (derived
from G) can pair with U, C or A.
As a result of the wobble, six codons for leucine require
only 3 tRNAs (2 shown below).

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Proteins General Information on R Groups

Strongly Acidic or Basic

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Weakly Acidic or Basic

Proteins General Information on R Groups

Hydrophobic

Gly=flexible; Cys=disulfide; Pro=hydrophobic ring

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Snapshots of TRANSLATION
Translation: Protein synthesis.
Complex anabolic activity involving:

mRNAs.
tRNAs with appropriate AAs.
Ribosomes.
Numerous proteins of specific functions.

3 distinct activities: Initiation, elongation,

termination.
http://www.youtube.com/watch?v=5bLEDd-PSTQ Translation

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Initiation
Ribosome attaches to precise site called initiation
codon (AUG) of mRNA.
The ribosome is now said to be in proper reading
frame: it correctly reads the entire message from
that point on.
Ribosome moves along and read from one codon
to the next, i.e. in consecutive 3 nucleotides.
For example, the ribosome moves from initiation
codon, then to the next codon, UCA, then GCU,
and so on along the entire line.
--- CUAUGUCAGCUCGGCUGCU --38

Process of Translation

https://www.google.com.sg

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Termination
3 stop codons (UAA, UAG, UGA) terminate pp assembly
and release pp associated with last tRNA.
Requires codon-recognizing Release Factors (RF). In
prokaryotic cells:
RF1 (recognizes UAA and UAG)
RF2 (recognizes UAA and UGA)
RF3 (non-codon specific and enhance activity of other factors)

RFs enter the A site, hydrolysis of ester bond linking the pp

chain to the tRNA, releasing the pp.
Ribosome disassembles for next round of translation

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Intracellular Organelles:
endoplasmic reticulum (ER)

Rough ER
- ribosomes attached
- protein secreting
- site of biosynthesis

Smooth ER
- lacks ribosomes
- synthesis of hormone
- release of glucose from
glycogen
- sequesting Ca2+

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Intracellular Organelles: Rough ER

Synthesis of Secretory Proteins

- mRNA binds to free ribosome
- N-terminal signal peptide synthesized and target new protein to ER
- recognition of SRP bound protein by SRP receptor
- recognition of ribosome and membrane channel (BIP)
- SRP released, protein synthesis resumed and channel widened
- protein released in ER lumen, and ribosome released for reuse
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Intracellular Organelles: Rough ER

Synthesis of Integral Membrane Proteins

- same initial event in secretory protein till recognition by BIP
- translocation of newly synthesized integral protein in channel stopped by
transmembrane helice
- channel opened and integral protein transferred to lipid bilayer

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Intracellular Organelles: Rough ER

Membrane Biosynthesis
- continued process by fusing with
existing membrane
- membrane components move to
different compartments (ER, Golgi) and
receive modifications
- membrane components show
asymmetry (glycosylation)

Synthesis of Membrane Lipids

- membrane lipids are made in ER and
Golgi
- phospholipids are made by ER
membrane enzymes
- membrane lipids inserted to plasma
membrane together with other
components
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Protein Folding and Synthesis in

Cell

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Protein Folding in Cell: Chaperones

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Protein Folding: Mechanism of

Chaperones

https://www.youtube.com/watch?v=d1QIEQEyYRo

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Summary
Translation
Initiation
Elongation
Termination
Translation in Cytosol and RER
Protein Folding and Activity

https://www.youtube.com/watch?v=--NcNeLc1mo Protein Folding

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Tutorial Topic
Wobble Hypothesis
Open Reading Frames: from DNA to
Protein
Frame 1: UCU AAA AUG GGU GAC
Frame 2: ...CUA AAA UGG GUG AC
Frame 3: .....UAA AAU GGG UGA C

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