GENE EXPRESSION: TRANSCRIPTION & TRANSLATION

Indwiani Astuti Dept of Pharmacology & Therapy Fac of Medicine Universitas Gadjah Mada

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DNA and Gene Expression

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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

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What are the bases in DNA

A adenine T thymine C cytosine G guanine Base pair rules

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Where is DNA located in the Cell?

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Where are the genes located?

Genes are located on the chromosomes. Every species has a different number of chromosomes. There are two types of chromosomes: autosomes and sex chromosomes
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 Genes are located on the chromosomes which are found in the nucleus of a cell. When a cell is undergoing cell reproduction, the chromosomes are visible. Chromosomes appear 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 information contained in an individual.
(gene + chromosome)
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What is gene expression?

Gene expression is the activation of a gene that results in a protein. Biological processes, such as transcription, and in case of proteins, also translation, that yield a gene product. A gene is expressed when its biological product is present and active. Gene expression is regulated at multiple levels.
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DNA is embedded in chromatin

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Regulation of gene expression at the level of chromatin

Sequence-independent

linker histones: control DNA compaction and accessibility to trans-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

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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.

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Eukaryotic Chromosome Structure

Methylation (the addition of CH3) 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.
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Regulation of gene expression

Promoter Gene (red) with an intron (green) single copy vs. multicopy plasmids 1. DNA replication Plasmid 2. Transcription Primary transcript 3. Posttranscriptional processing 4. Translation inactive protein 5. Posttranslational processing active protein indwiani@gmail.com Mature mRNA mRNA degradation

Protein degradation

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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.

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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

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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.

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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 in function nucleus, mitochondria, chloroplast) Chromosomes are in pairs and not circular All organisms that are not bacteria: protist, fungi, plants and animals
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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 main classes 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 into the amino acid sequence of a protein using ribosomes, large complexes of rRNAs and proteins.
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Proteins Regulate Gene Expression

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Gene structure relevant to metabolic regulation

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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 using the genetic code on the mRNA molecule as its guide).
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 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 regions that do not code for proteins are called INTRONS.

cytoplasm cytoplasm

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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.

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Intron Splicing in Eukaryotes

Exons: coding regions Introns: noncoding regions Introns are removed by splicing

GU at 5 end of intron

AG at 3 end of intron

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Splicesomes Roles in Spicing out Intron

RNA splicing occurs in small nuclear ribonucleoprotein particles (snRNPS) in spliceosomes

Spliceosomes: protein + small RNAs (U1-8) complementar y to the splice junctions

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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 transcription apparatus

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Enhancer Control
Eukaryote genes on a DNA strand 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 with transcription.

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 In prokayotes, transcription and translation occur in the cytoplasm. In eukaryotes, transcription occurs inside the nucleus in a two step sequence of events.
Pre-mRNA includes both introns and exons for the gene. mRNA is only the coding portion (exons).

Translation occurs in the cytoplasm at the ribosomes.

Reminder: The are three (3) types of RNA
Messenager (mRNA) Transfer (tRNA) Ribsomal (rRNA)
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RIBONUCLEIC ACID (RNA)

Found all over the cell (nucleus, mitochondria, chloroplasts, ribosomes and the soluble part of the cytoplasm)

2007 Paul Billiet ODWS

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Types
Messenger RNA (mRNA) <5% Ribosomal RNA (rRNA) Up to 80% Transfer RNA (tRNA) About 15% In eukaryotes small nuclear ribonucleoproteins (snRNP)

2007 Paul Billiet ODWS

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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).

2007 Paul Billiet ODWS

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mRNA
A long molecule 1 million Daltons Ephemeral Difficult to isolate mRNA provides the plan for the polypeptide chain

2007 Paul Billiet ODWS

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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
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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

2007 Paul Billiet ODWS

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Transcription: The synthesis of a strand of mRNA (and other RNAs)

Uses an enzyme RNA polymerase Proceeds in the same direction as replication (5 to 3) Forms a complementary strand of mRNA It begins at a promotor site which signals the beginning of gene is not much further down the molecule (about 20 to 30 nucleotides) After the end of the gene is reached there is a terminator sequence that tells RNA polymerase to stop transcribing NB Terminator sequence terminator codon
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Editing the mRNA

In prokaryotes the transcribed mRNA goes straight to the ribosomes in the cytoplasm In eukaryotes the freshly transcribed mRNA in the nucleus is about 5000 nucleotides long When the same mRNA is used for translation at the ribosome 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
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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
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Posttranscriptional Regulation
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 mRNAs before translation

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Posttranscriptional Regulation
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.

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Posttranscriptional Regulation
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

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Posttranscriptional Regulation
Mature mRNA molecules have various halflives depending on the gene and the location (tissue) of expression. The amount of polypeptide produced from a particular gene can be influenced by the halflife of the mRNA molecules.

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Translation
RNA
Single stranded Does not contain thymine but has uracil instead.

tRNA carries 3 base pair code for specific amino acid. Amino acids compose polypeptid chains. One or more polypeptide chains compose a protein proteins provide the blueprints for our characteristics and functions.

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Transcription plan
Nucleus Gene

DNA Transcription messenger RNA

2007 Paul Billiet ODWS

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Translation plan
Complete protein Polypeptide chain TRANSLATION

Ribosomes

Stop codon
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Start codon

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SKEMA TRANSLASI

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Translation
Location: The ribosomes in the cytoplasm that provide the environment for translation The genetic code is brought by the mRNA molecule

2007 Paul Billiet ODWS

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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

2007 Paul Billiet ODWS

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How many combinations?

If one base represented one amino acid this would only be able to produce 4 different combinations. (A, C, G and U) If pairs of bases represented each amino acid this would only be able to produce 4 x 4 = 16 combinations. (AA, AC, AG, AU, CA, CC, CG, CU etc) If triplets of bases represented each amino acid, this would be able to produce 4 x 4 x 4 = 64 combinations

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

2007 Paul Billiet ODWS

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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
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Ribosomes initiate translation at ribosome-binding sites in polycistronic procaryotic mRNAs, which can encode more than one protein

07_33_mRNA.encode.jpg

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A polyribosome 07_35_polyribosome.jpg from a eucaryotic cell

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Initiation of Translation in Eukaryotes

Cap 5UTR AUG----------------Stop 3UTR AAAAAAAAA

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 indwiani@gmail.com

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Proteins
Essential for all biological events

DNA carries the information Protein does the business

Enormous diversity
functional structural

Simple building-blocks L-amino acids

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 Kuliah Selanjutnya Protein Selamat Belajar

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