gene es

7/30/2019 gene es

1/63

GENE EXPRESSION:

TRANSCRIPTION & TRANSLATIONIndwiani Astuti

Dept of Pharmacology & Therapy

Fac of MedicineUniversitas Gadjah Mada

[email protected]

7/30/2019 gene es

2/63

DNA and Gene Expression

[email protected]

7/30/2019 gene es

3/63

DNA

Deoxyribonucleic Acid

Double helix

Carries genetic information

Located in the nucleus

The monomer is a nucleotide

A phosphate

A ribose sugar A nitrogenous base

[email protected]

7/30/2019 gene es

4/63

What are the bases in DNA

A adenine

T thymine

C cytosine

G guanine

Base pair rules

[email protected]

7/30/2019 gene es

5/63

Where is DNA located in the Cell?

[email protected]

7/30/2019 gene es

6/63

Where are the genes located?

Genes are located on

the chromosomes.

Every species has a

different number ofchromosomes.

There are two types of

chromosomes:

autosomes and sex

chromosomes

[email protected]

7/30/2019 gene es

7/63

Genes are located on the chromosomes whichare found in the nucleus of a cell.

When a cell is undergoing cell reproduction,the chromosomes are visible. Chromosomesappear when the chromatin condenses and

become visible. Most of the time (90%) the genetic material in

the form of chromatin.

A genome is the complete genetic informationcontained in an individual.

(gene + chromosome)

[email protected]

7/30/2019 gene es

8/63

What is gene expression?

Gene expression is the activation of a gene thatresults in a protein.

Biological processes, such as transcription, and in

case of proteins, also translation, that yield agene product.

A gene is expressed when its biological product is

present and active.

Gene expression is regulated at multiple levels.

[email protected]

7/30/2019 gene es

9/63

[email protected]

7/30/2019 gene es

10/63

DNA is embedded in chromatin

[email protected]

7/30/2019 gene es

11/63

Regulation of gene expression at the level of chromatin

Sequence-independent

linker histones: control DNA compaction and accessibility totrans

-acting factors

post-translational modifications of histone tails: control compaction of DNA and

serve as docking sites for trans-acting factors

Range: Can act at the level of a single gene, often acts over groups of genes and

over larger domains (20-200kb), and can affect gene expression over an entire

chromosome

[email protected]

7/30/2019 gene es

12/63

12

Eukaryotic Chromosome Structure

Eukaryotic DNA is packaged into chromatin.

Chromatin structure is directly related to the

control of gene expression.

Chromatin structure begins with the

organization of the DNA into nucleosomes.

Nucleosomes may block RNA polymerase II

from gaining access to promoters.

[email protected]

7/30/2019 gene es

13/63

13

Eukaryotic Chromosome Structure

Methylation (the addition ofCH3) of DNA or

histone proteins is associated with the control

of gene expression.

Clusters of methylated cytosine nucleotides

bind to a protein that prevents activators from

binding to DNA.

Methylated histone proteins are associated

with inactive regions of chromatin.

[email protected]

7/30/2019 gene es

14/63

[email protected]

7/30/2019 gene es

15/63

[email protected]

7/30/2019 gene es

16/63

Regulation of gene expression

Plasmid

Gene (red) with an intron (green)Promoter

2.Transcription

Primary

transcript

1. DNA replication

3. Posttranscriptionalprocessing

4. Translation

mRNA degradation

Mature

mRNA

5. Posttranslationalprocessing

Protein degradationinactiveprotein

active

protein

single copy vs. multicopy plasmids

[email protected]

7/30/2019 gene es

17/63

17

Control of Gene Expression

Controlling gene expression is often

accomplished by controlling transcription

initiation.

Regulatory proteins bind to DNA to either

block or stimulate transcription, depending on

how they interact with RNA polymerase.

[email protected]

7/30/2019 gene es

18/63

Gene expression regulation at the level of DNA

Sequence-dependent

cis-acting factors: promoters/regulatory sequences of genes

trans-acting factors: proteins and RNAs that bind cis-elements and promote or

repress gene expression

DNA methylation: methylation of CpG islands promotes silencing

Range: Usually functions at level of single gene, or at most a local group of genes

[email protected]

7/30/2019 gene es

19/63

19

Control of Gene Expression

Prokaryotic organisms regulate gene

expression in response to their environment.

Eukaryotic cells regulate gene expression to

maintain homeostasis in the organism.

[email protected]

7/30/2019 gene es

20/63

Gene expression takes place differently in

prokaryotes and eukaryotes. What is a

prokaryotes? Eukaryotes?

Prokaryotes

No membrane bound

organelles (nucleus) More primitive

organisms

Only one circular

chromosome Bacteria are the only

organisms that are

prokaryotes.

Eukaryotes

Membrane bound

organelles ( specialize infunctionnucleus,

mitochondria, chloroplast)

Chromosomes are in pairs

and not circular All organisms that are not

bacteria: protist, fungi,

plants and animals

[email protected]

7/30/2019 gene es

21/63

21

Expression of Genetic Information

Production of proteins requires two steps: Transcription involves an enzyme (RNA polymerase) making an

RNA copy of part of one DNA strand. There are four mainclasses of RNA:

i. Messenger RNAs (mRNA), which specify the amino acid

sequence of a protein by using codons of the genetic code.ii. Transfer RNAs (tRNA).

iii. Ribosomal RNAs (rRNA).

iv. Small nuclear RNAs (snRNA), found only in eukaryotes.

Translation converts the information in mRNA intothe amino acid sequence of a protein usingribosomes, large complexes of rRNAs andproteins.

[email protected]

7/30/2019 gene es

22/63

22

Proteins Regulate Gene Expression

[email protected]

7/30/2019 gene es

23/63

23

Gene structure relevant to

metabolic regulation

[email protected]

In Eukaryotes following mitosis or meiosis DNA recoils

7/30/2019 gene es

24/63

In Eukaryotes, following mitosis or meiosis, DNA recoils

but certain regions remain relaxed for transcription.

The areas of relaxed DNA are called euchromatin.

Transcription is the

Reading of the DNA and

Changing the code to

mRNA.

Translation is changing

The mRNA into a trait by

Using tRNA to interpret the

mRNA (The synthesis of a

polypeptide chain usingthe genetic code on themRNA molecule as itsguide).

[email protected]

7/30/2019 gene es

25/63

DNA in eukaryotes has regions of coding and

noncoding DNA. The regions of DNA that code for

proteins or traits are called EXONS, while the regionsthat do not code for proteins are calledINTRONS.

cytoplasmcytoplasm

[email protected]

7/30/2019 gene es

26/63

26

Eukaryotic Transcription

General transcription factors bind to the

promoter region of the gene.

RNA polymerase II then binds to the promoter

to begin transcription at the start site (+1).

Enhancers are DNA sequences to which

specific transcription factors (activators) bind

to increase the rate of transcription.

[email protected]

7/30/2019 gene es

27/63

27

Exons: coding regions

Introns:noncoding regions

Introns are removed by splicing

GU

at 5 endof intron

AG

at 3 endof intron

[email protected]

7/30/2019 gene es

28/63

28

RNA splicing occurs in small nuclear ribonucleoproteinparticles (snRNPS) in spliceosomes

Spliceosomes:

protein +small RNAs(U1-8)complementary to thesplice

junctions

[email protected]

7/30/2019 gene es

29/63

[email protected]

7/30/2019 gene es

30/63

30

Eukaryotic Transcription

Coactivators and mediators are also required

for the function of transcription factors.

coactivators and mediators bind to transcription

factors and bind to other parts of the transcriptionapparatus

[email protected]

7/30/2019 gene es

31/63

[email protected]

7/30/2019 gene es

32/63

Enhancer Control

Eukaryote genes on a DNAstrand also have noncoding

control sequences that

facilitate transcription.

These are called enhancers.

Transcription factors are

additional proteins that

bind to RNA polymerase

and enhancers to help withtranscription.

[email protected]

7/30/2019 gene es

33/63

In prokayotes, transcription and translationoccur in the cytoplasm.

In eukaryotes, transcription occurs inside thenucleus in a two step sequence of events.

Pre-mRNA includes both introns and exons for thegene.

mRNA is only the coding portion (exons).

Translation occurs in the cytoplasm at theribosomes.

Reminder: The are three (3) types of RNA Messenager (mRNA)

Transfer (tRNA)

Ribsomal (rRNA)

[email protected]

7/30/2019 gene es

34/63

RIBONUCLEIC ACID (RNA)

Found all over the cell (nucleus, mitochondria,

chloroplasts, ribosomes and the soluble part of

the cytoplasm)

2007 Paul Billiet ODWS [email protected]
http://www.saburchill.com/IBbiology/bio_hp.htmlhttp://www.saburchill.com/IBbiology/bio_hp.html

7/30/2019 gene es

35/63

Types

Messenger RNA (mRNA)

7/30/2019 gene es

36/63

Structural characteristics of RNA

molecules

Single polynucleotide strand which may be

looped or coiled (not a double helix).

Sugar Ribose (not deoxyribose).

Bases used: Adenine, Guanine, Cytosine and

Uracil(not Thymine).


7/30/2019 gene es

37/63

mRNA

A long molecule 1 million Daltons

Ephemeral

Difficult to isolate

mRNA provides the plan for the polypeptide

chain


7/30/2019 gene es

38/63

rRNA

Coiled

Two subunits:

a long molecule 1 million Daltons

a short molecule 42 000 Daltons

Fairly stable

Found in ribosomes

Made as subunits in the nucleolus

rRNA provides the platform from protein

synthesis 2007 Paul Billiet ODWS [email protected]

7/30/2019 gene es

39/63

tRNA

Short molecule about 25 000 Daltons

Soluble

At least 61 different forms each has a specific

anticodon as part of its structure.

tRNA translates the message on the mRNA

into a polypeptide chain


7/30/2019 gene es

40/63

Transcription: The synthesis of a strand of

mRNA (and other RNAs)

Uses an enzyme RNA polymerase

Proceeds in the same direction as replication (5 to3)

Forms a complementary strand of mRNA It begins at a promotor site which signals the

beginning of gene is not much further down themolecule (about 20 to 30 nucleotides)

After the end of the gene is reached there is aterminator sequence that tells RNA polymerase tostop transcribing

NB Terminator sequence terminator codon 2007 Paul Billiet ODWS

[email protected]

7/30/2019 gene es

41/63

Editing the mRNA

In prokaryotes the transcribed mRNA goes straightto the ribosomes in the cytoplasm

In eukaryotes the freshly transcribed mRNA in thenucleus is about 5000 nucleotides long

When the same mRNA is used for translation at theribosome it is only 1000 nucleotides long

The mRNA has been edited

The parts which are kept for gene expression are

called EXONS (exons = expressed) The parts which are edited out (by snRNP molecules)

are called INTRONS

2007 Paul Billiet [email protected]

7/30/2019 gene es

42/63

42

Posttranscriptional Regulation

Control of gene expression usually involves the

control of transcription initiation.

But gene expression can be controlled after

transcription, with mechanisms such as:

RNA interference

alternative splicing

RNA editing

mRNA degradation

[email protected]

7/30/2019 gene es

43/63

43


RNA interference involves the use of small

RNA molecules

The enzyme Dicer chops double stranded RNA

into small pieces of RNA

micro-RNAs bind to complementary RNA to

prevent translation

small interfering RNAs degrade particular mRNAsbefore translation

[email protected]

7/30/2019 gene es

44/63

44


Introns are spliced out of pre-mRNAs to produce

the mature mRNA that is translated.

Alternative splicing recognizes different splice

sites in different tissue types.

The mature mRNAs in each tissue possess

different exons, resulting in different

polypeptide products from the same gene.

[email protected]

7/30/2019 gene es

45/63

45


RNA editing creates mature mRNA that are

not truly encoded by the genome.

For example

apolipoprotein B exists in 2 isoforms

one isoform is produced by editing the mRNA to

create a stop codon

this RNA editing is tissue-specific

[email protected]

7/30/2019 gene es

46/63

46


Mature mRNA molecules have various half-

lives depending on the gene and the location

(tissue) of expression.

The amount of polypeptide produced from a

particular gene can be influenced by the half-

life of the mRNA molecules.

[email protected]

7/30/2019 gene es

47/63

[email protected]

l i

7/30/2019 gene es

48/63

Translation RNA

Single stranded

Does not contain thymine buthas uracil instead.

tRNA carries 3 base pair codefor specific amino acid.

Amino acids composepolypeptid chains.

One or more polypeptidechains compose a protein

proteins provide the

blueprints for ourcharacteristics and functions.

[email protected]

7/30/2019 gene es

49/63

Transcription plan

Transcription

DNA

messengerRNA

Gene

Nucleus


7/30/2019 gene es

50/63

Translation plan

TRANSLATION

Complete protein

Polypeptide chain

Ribosomes

Stop codon Start codon


7/30/2019 gene es

51/63

SKEMA TRANSLASI

[email protected]

7/30/2019 gene es

52/63

Translation

Location: The ribosomes in the cytoplasm that

provide the environment for translation

The genetic code is brought by the mRNA

molecule


7/30/2019 gene es

53/63

What is the genetic code?

The genetic code consists of the sequence of

bases found along the mRNA molecule

There are only four letters to this code (A, G, C

and U)

The code needs to be complex enough to

represent 20 different amino acids used to

build proteins


7/30/2019 gene es

54/63

[email protected]

7/30/2019 gene es

55/63

[email protected]

7/30/2019 gene es

56/63

How many combinations?

Ifone base represented one amino acid this would only beable to produce

4 different combinations. (A, C, G and U)

Ifpairsof bases represented each amino acid this would onlybe able to produce

4 x 4 = 16 combinations. (AA, AC, AG, AU, CA, CC, CG, CU etc)

Iftriplets of bases represented each amino acid, this wouldbe able to produce

4 x 4 x 4 = 64 combinations

This is enough combinations to code for the 20 amino acidsbut is the code actually made of triplets?

2007 Paul Billiet [email protected]

7/30/2019 gene es

57/63

Nature is logical!

Over 10 years biochemists synthesised bits of

mRNA with different combinations

Then they used them to synthesise

polypeptides

The results proved the logical answer was

correct

The genetic code is made of triplets of bases

called codons


7/30/2019 gene es

58/63

07_33_mRNA.encode.jpgRibosomes initiate translation at ribosome-binding sitesin polycistronic procaryotic mRNAs, which can encodemore than one protein

[email protected]

7/30/2019 gene es

59/63

07_35_polyribosome.jpgA polyribosome from aeucaryotic cell

[email protected]

Initiation of Translation in E kar otes

7/30/2019 gene es

60/63

Initiation of Translation in Eukaryotes

AAAAAAAAACapAUG----------------Stop

5UTR 3UTR

Important points:

No direct binding between mRNA and rRNA

Small ribosome subunit binds directly to cap requires

specific initiation factor eIF4e

Other initiation factors can unwind double stranded regions

in the mRNA eIF4 group

Small subunit scans mRNA till it finds correct AUG

Correct AUG is embedded in preferred sequence

GccAccAUGG

G [email protected]

7/30/2019 gene es

61/63

Proteins Essential for all biological events

DNA carries the information

Protein does the business

Enormous diversity functional

structural

Simple building-blocks - L-amino acids

[email protected]

7/30/2019 gene es

62/63

[email protected]

7/30/2019 gene es

63/63

Kuliah Selanjutnya Protein

Selamat Belajar

gene es

Documents