How Cells Read the Genome: From DNA to Protein M. Saifur Rohman, Sp.JP.,Ph.D.

How Cells Read the Genome: From DNA

to Protein

M. Saifur Rohman, Sp.JP.,Ph.D.

An Overview of Gene Control

• A chromosome is an organized building of DNA and protein that is found in cells

• A single piece of coiled DNA containing many genes, regulatory elements and other nucleotide sequences.

• Chromosomes also contain DNA-bound proteins, which serve to package the DNA and control its functions.

• Chromosomes vary widely between different organisms.

Chromosome

The DNA molecule may be circular or linear, and can be composed of 10,000 to 1,000,000,000 nucleotides in a long chain.

In eukaryotes, nuclear chromosomes are packaged by proteins into a condensed structure called chromatin.

Chromosome

• This allows the very long DNA molecules to fit into the cell nucleus.

• Chromosomes are the essential unit for cellular division and must be replicated, divided, and passed successfully to their daughter cells so as to ensure the genetic diversity and survival of their progeny.

Chromosome

If these structures are manipulated incorrectly, through processes known as chromosomal instability and translocation, the cell may undergo mitotic catastrophe and die, or it may aberrantly evade apoptosis leading to the progression of cancer.

In prokaryotes DNA is usually arranged as a circle, which is tightly coiled in on itself, sometimes accompanied by one or more smaller, circular DNA molecules called plasmids.

Chromosome

• Chromosomes can be divided into two types--autosomes, and sex chromosomes.

• Certain genetic traits are linked to your sex, and are passed on through the sex chromosomes.

• The autosomes contain the rest of the genetic hereditary information. All act in the same way during cell division.

• Human cells have 23 pairs of large linear nuclear chromosomes, (22 pairs of autosomes and one pair of sex chromosomes) giving a total of 46 per cell.

Chromosome

• Human cells have many hundreds of copies of the mitochondrial genome.

• Sequencing of the human genome has provided a great deal of information about each of the chromosomes

• Number of genes is an estimate as it is in part based on gene predictions.

• Total chromosome length is an estimate as well, based on the estimated size of unsequenced heterochromatin regions.

Chromosome

• The set of chromosomes in a cell makes up its genome; the human genome has approximately 3 billion base pairs of DNA arranged into 46 chromosomes.[

• The information carried by DNA is held in the sequence of pieces of DNA called genes.

• For example, in transcription, when a cell uses the information in a gene, the DNA sequence is copied into a complementary RNA sequence through the attraction between the DNA and the correct RNA nucleotides.

Gene

• A cell may simply copy its genetic information in a process called DNA replication.

• Genomic DNA is tightly and orderly packed in the process called DNA condensation to fit the small available volumes of the cell.

• The genetic information in a genome is held within genes, and the complete set of this information in an organism is called its genotype

Genome

• A gene is a unit of heredity and is a region of DNA that influences a particular characteristic in an organism.

• Genes contain an open reading frame that can be transcribed, as well as regulatory sequences such as promoters and enhancers, which control the transcription of the open reading frame.

• In many species, only a small fraction of the total sequence of the genome encodes protein.

Regulatory seq. of gene

Only about 1.5% of the human genome consists of protein-coding exons, with over 50% of human DNA consisting of non-coding repetitive sequences.

The reasons for the presence of so much non-coding DNA in eukaryotic genomes and the extraordinary differences in genome size, or C-value, among species represent a long-standing puzzle known as the "C-value enigma".

However, DNA sequences that do not code protein may still encode functional non-coding RNA molecules, which are involved in the regulation

of gene expression.

Coding vs. non coding

Review : DNA in Chromosome

DNA in chromosome

• Within cells, DNA is organized into long structures called chromosomes.

• These chromosomes are duplicated before cells divide, in a process called DNA replication.

• Eukaryotic organisms(animals plants, fungi, and protists) store most of their DNA inside the cell nucleus and some of their DNA in organelles such as mitochondria or chloroplasts

DNA

• Although each individual repeating unit is very small, DNA polymers can be very large molecules containing millions of nucleotides.

• The largest human chromosome, chromosome number 1, is approximately 220 million base pairs long.

• In living organisms, DNA does not usually exist as a single molecule, but instead as a pair of molecules that are held tightly together.

From DNA to Protein

Transcription

Transcription Steps

From DNA to RNA

Transcription regulation

RNA Polymerase II

Transcription Factor

Promoter and Enhancer

TATA Binding Protein

A Transcription Factor Complex

On- off Histone regulation

Histone Deacetylation

mRNA Processing

Splicing mRNA

tRNA

Ribosomal RNA

Initiation factors

From nucleotides to amino acid

Transcription

Post transcription Control

• Processing of eukaryotic pre-mRNA- capping - polyadenylation- splicing- editing• Nuclear transport

Post transcriptional controls• Post-initiation transcriptional control of gene expression

• attenuation• Alternative splicing

• Regulation of alternative splicing

• Transcript cleavage • Secreted verses membrane bound antibodies

• RNA editing especially as it related to human cells• RNA transport and localization

• Export of HIV RNAs from the nucleus• Localization in the cytoplasm

• Negative control of translation initiation• Bacteria (ex. Bacterial ribosomal proteins)• How do translational repressor work in eukaryotes

–Aconitase• Phosphorylation of eIF-2• uORFs

• IRES• Control of mRNA stability• RNA interference, miRNAs, siRNAs

Processing of eukaryotic pre-mRNA: the classical texbook picture

Alternative picture: co-transcriptional pre-mRNA processing

• This picture is more realistic than the previous one, particularly for long pre-mRNAs

Splicing

Small nuclear RNAs U1-U6 participate in splicing

• snRNAs U1, U2, U4, U5 and U6 form complexes with 6-10 proteins each, forming small nuclear ribonucleoprotein particles (snRNPs)

• Sm- binding sites for snRNP proteins

Binding of U1 and U2 snRNPs

Binding of U4, U5 and U6 snRNPs

The essential steps in splicing

Spliceosome• Spliceosome contains snRNAs, snRNPs and many

other proteins, totally about 300 subunits. • This makes it the most complicted macromolecular

machine known to date.• But why is spliceosome so extremely complicated if

it only catalyzes such a straightforward reaction as an intron deletion? Even more, it seems that some introns are capable to excise themselves without aid of any protein, so why have all those 300 subunits?

RNA editing

• Enzymatic altering of pre-mRNA sequence• Common in mitochondria of protozoans and plants and

chloroplasts, where more than 50% of bases can be altered

• Much rarer in higher eukaryotes

Editing of human apoB pre-mRNA

The two types of editing1) Substitution editing• Chemical altering of individual nucleotides• Examples: Deamination of C to U or A to I

(inosine, read as G by ribosome)

2) Insertion/deletion editing• Deletion/insertion of nucleotides (mostly uridines) • For this process, special guide RNAs (gRNAs) are

required

Guide RNAs (gRNAs) are required for editing

Assembly of ribosomes

Processing of pre-tRNAs

RNase P cleavage site

Splicing of pre-tRNAs is different from pre-mRNAs and pre-rRNAs

• The splicing of pre-tRNAs is catalyzed by protein only

• A pre-tRNA intron is excised in one step, not by two transesterification reactions

• Hydrolysis of GTP and ATP is required to join the two RNA halves

Macromolecular transport across the nuclear envelope

The central channel

• Small metabolites, ions and globular proteins up to ~60 kDa can diffuse freely through the channel

• Large proteins and ribonucleoprotein complexes (including mRNAs) are selectively transported with the assistance of transporter proteins

Two different kinds of nuclear location sequences basic

hydrophobic

importin a importin b

importin b

nuclear import

Proteins which are transported into nucleus contain nuclear location sequences

Mechanism for nuclear “import”

Mechanism for nuclear “export”

Mechanism for mRNA transport to cytoplasm

Example of regulation at nuclear transport level: HIV mRNAs

After mRNA reaches the cytoplasm...

• mRNA exporter, mRNP proteins, nuclear cap-binding complex and nuclear poly-A binding proteins dissociate from mRNA and gets back to nucleus

• 5’ cap binds to translation factor eIF4E• Cytoplasmic poly-A binding protein (PABPI) binds

to poly-A tail• Translation factor eIF4G binds to both eIF4E and

PABPI, thus linking together 5’ and 3’ ends of mRNA

The control of gene expression

• Each cell in the human contains all the genetic material for the growth and development of a human

• Some of these genes will be need to be expressed all the time

• These are the genes that are involved in of vital biochemical processes such as respiration

• Other genes are not expressed all the time• They are switched on an off at need

© 2007 Paul Billiet ODWS

On-Off Gene Activation

How Genetic Switches Work

Carbohydrates Activator protein

Repressor protein

RNA polymerase

lac Operon

+ GLUCOSE+ LACTOSE

Not bound to DNA

Lifted off operator site

Keeps falling off promoter

site

No transcription

+ GLUCOSE- LACTOSE

Not bound to DNA

Bound to operator site

Blocked by the repressor

No transcription

- GLUCOSE- LACTOSE

Bound to DNA

Bound to operator site

Blocked by the repressor

No transcription

- GLUCOSE+ LACTOSE

Bound to DNA

Lifted off operator site

Sits on the promoter site

Transcription

© 2007 Paul Billiet ODWS

DNA-Binding Motifs in Gene Regulatory Proteins

DNA-Binding Motifs in Gene Regulatory Proteins

Translation

Translation - animation

mRNA attaches to small ribosomal subunit

Translation - outline

Translation. mRNA used to make polypeptide chain (protein)

1.

•First the mRNA attaches itself to a ribosome (to the small subunit).•Six bases of the mRNA are exposed.•A complementary tRNA molecule with its attached amino acid (methionine) base pairs via its anticodon UAC with the AUG on the mRNA in the first position P.•Another tRNA base pairs with the other three mRNA bases in the ribosome at position A.•The enzyme peptidyl transferase forms a peptide bond between the two amino acids.•The first tRNA (without its amino acid) leaves the ribosome.

Translation 2

The ribosome moves along the mRNA to the next codon (three bases).The second tRNA molecule moves into position P.Another tRNA molecule pairs with the mRNA in position A bringing its amino acid.A growing polypeptide is formed in this way until a stop codon is reached.

End of Translation

A stop codon on the mRNA is reached and this signals the ribosome to leave the mRNA. A newly synthesised protein is now complete!

TranslationmRNA to Polypeptide

rNA to Protein

• Multi steps – complex system

Example of expression regulation

mPer2 conditional knock out construct

loxlox FLPFLP

SacI-blSalI

SalIKpnI-bl

Intron2 Exon2 DT-A

Pgk-neo

Stem cell selection and implantation

X

NHE3

?

CACGTG GGATCC

BMAL1 Clock

NHE3 ConstructD

?

NHE3 activation through E-box ?

BMAL1 Clock

NHE3

?

Method: In vitro Transcription Assay

Method: Site Direct Mutagenesis

How genome evolve ?

DNA Replication and Repair

DNA Replication…

• Reproduction is fundamental to all living systems • Regardless of the reproductive mechanism (asexual

or sexual) a method must exist to transfer genetic material from one generation to the next.

• DNA must be copied (replicated) in a manner that minimizes mistakes.

• Damage to DNA must be repaired to prevent that damage from being transferred to the next generation.

– Replication occurred by gradual double helix strand separation via successive breakage of H bonds, much like the separation of the two halves of a zipper

– Since each strand is complementary to the other, each has the information needed to construct the other; once separated, each strand can serve as template to direct the formation of the other strand

DNA Replication…

• Possible types of DNA replication – 1. Semiconservative - daughter

duplex made of one parental & one newly synthesized strand

– 2. Conservative - 2 original strands stay together after serving as templates for 2 new strands that also stay together; one contains only "old" DNA, the other only "new" DNA

– 3. Dispersive – integrity of both parental strands disrupted; new duplex strands made of old & new DNA; neither the parental strands nor the parental duplex is preserved

DNA Replication…

• DNA is very susceptible to environmental damage • A. Types of damage experienced by DNA

– 1. Ionizing radiation can break DNA backbone – 2. Exposure to a variety of reactive chemicals can alter DNA bases– 3. Ultraviolet radiation causes adjacent pyrimidines (C or T) to interact

covalently– 4. Thermal energy generated by metabolism in a warm-blooded bird

or mammal can split adenine & guanine from their attachment to DNA backbone sugars

• B. Spontaneous alterations or lesions occur often– Each cell of a warm-blooded mammal loses ~10,000 bases/day

DNA Repair…

• DNA damage… • Potential effects

– Gametes – If damage occurs in a cell destined to become a gamete, the damage (mutation) can be passed on and become a permanent part of the population’s gene pool

– Somatic cells – not possible to pass the mutation on but…• 1. Can interfere with transcription and replication • 2. Can lead to the malignant transformation of a cell • 3. Can speed the process by which an organism ages

– It is vital that cells have a way to repair the damage and they do• Damage is kept to <1 nucleotide/1000 bases

DNA Repair…

• DNA Repair: Nucleotide Excision Repair (NER) • Removes part of strands having lesions:

– pyrimidine dimers & chemically altered nucleotides; – "cut-and-patch" mechanism; – A. Transcription-coupled pathway - template strands of genes that

are being actively transcribed are preferentially repaired; repair of template strand is thought to occur as DNA is being transcribed

• 1. The presence of the lesion may be signaled by a stalled RNA polymerase

• 2. Ensures that those genes of greatest importance to the cell, the genes being actively transcribed, receive the highest priority on the repair list

– B. Global pathway - slower, less efficient pathway that corrects DNA strands in remainder of genome

DNA Repair…

• DNA Repair: Nucleotide Excision Repair (NER) • Steps in NER process in eukaryotic cells –

– 1. Lesion recognition by proteins scanning DNA recognize distorted sites in helix

– 2. The recruitment of repair enzymes to the lesion– 3. The damaged strand is cut on both sides of the lesion by a pair of

endonucleases; the segment of damaged DNA is now held in position only by H bonds

– 4. Segment of DNA between the incisions is released – 5. The gap is filled by DNA polymerase & the strand is sealed by DNA

ligase – See animation and movie on website

DNA Repair…

• DNA Repair: Base Excision Repair (BER) • Sometimes single nucleotides in the double helix are

altered via chemical reaction and become mismatched

• A. Uracil - forms by hydrolytic removal of cytosine's amino group

• B. 8-oxo-guanine - caused by damage from oxygen free radicals

• C. 3-methyladenine - caused by alkylating agents; transfer of methyl group from a methyl donor

DNA Repair…

• DNA Repair: Base Excision Repair (BER)

• Steps in BER process in eukaryotes – Initiated by a DNA

glycosylase that recognizes alteration

– DNA glycosylase removes the base (not the entire nucleotide)

DNA Repair…

• DNA Repair: Base Excision Repair (BER)

• Steps in BER process in eukaryotes – The "beheaded" deoxyribose

phosphate is removed by (AP) endonuclease & DNA polymerase

– 1. The AP endonuclease cleaves the DNA backbone

– 2. Polymerase β removes the sugar-phosphate remnant that had been attached to the excised base

– 3. Gap filled by DNA polymerase β – 4. Strand is sealed by DNA ligase

DNA Repair…

• DNA Repair: Mismatch Repair • Cells replace mismatched bases that are

incorporated by DNA polymerase & escape the enzyme's proofreading exonuclease

• Mismatched base pairs cause distortions in double helix geometry that are recognized by a repair enzyme

• Problem: How does repair system know which member of a mismatched pair is the incorrect nucleotide?

DNA Repair…

DNA Repair…• DNA Repair: Mismatch Repair • Cells rely on being able to

distinguish between new and old strands after replication

• Newly made strand contains the incorrect nucleotide; parental strand contains the correct one

• In prokaryotes, new and old strands are distinguished based on methylation

• How eukaryotes identify newly synthesized strands remains unclear

Thank You