Molecular Basis of Inheritance

Neeta Sehgal University of Delhi

GENE
Gene is a linear segment of DNA Some genes are used to direct the synthesis of RNA Deoxyribonucleic Acid (DNA) Ribonucleic Acid (RNA)

DNA
Double helix: Two polynucleotide chains wound around each other The helix twists in the right-handed direction i.e. clockwise direction.

Semiconservative Model of DNA Replication

Double-stranded DNA
5 3

Major groove Minor groove

B DNA
3 5 3 5

BioSynthesis of DNA
DNA
Nucleotides (Adenine, Thymine, Cytosine, Guanine) Deoxyribose sugar Phosphoric acid

Synthesis of DNA
DNA polymerase, dNTPs DNA strand as a template. 5' to 3' direction

DNA Replication
Replication of antiparallel DNA Strands
Double stranded DNA Two DNA strands are antiparallel, Replication fork 5' to 3' direction. DNA polymerase synthesizes new DNA A large number of enzymes

DNA polymerase catalyzes the synthesis of DNA using a DNA strand as a template.
The DNA unwounds to form a replication fork. On the bottom template strand, primase synthesizes a short RNA primer in the 5' to 3' direction. Primase leaves, and DNA polymerase adds DNA nucleotides to the RNA primer in the 5' to 3' direction. Newly syntheiszed DNA is called the leading strand

leading strand

On the top template strand, primase synthesizes a short RNA primer in the 5' to 3' direction. Primase leaves, and DNA polymerase adds DNA nucleotides to the RNA primer in the 5' to 3' direction. This new DNA is called the lagging strand because it is being made in the direction opposite to the movement of the replication fork. The segment produced is also called an Okazaki fragment. More DNA unwinds Leading strand is extended by DNA polymerase adding more DNA nucleotides. Thus, the leading strand is synthesized continuously. On the top template strand, a new RNA primer is synthesized by primase near the replication fork DNA is added to it by DNA polymerase. Second Okazaki fragment is produced. the lagging strand is synthesized discontinously. DNA ligase joins the two Okazaki fragments to produce a continuous chain. The process repeats as the DNA continues to unwind.

New DNA strand is synthesized continuously and the other is synthesized discontinuously, this model is called the semidiscontinuous model for DNA synthesis

A different type of DNA polymerase removes the RNA primer and replaces it with DNA. DNA ligase joins the two Okazaki fragments to produce a continuous chain.

Semidiscontinuous model for DNA synthesis

DNA continues to unwind. New DNA strand is synthesized continuously The other is synthesized discontinuously

Gene Expression
The information is on DNA Decoding the information on gene for a specific protein This first step of gene expression is called Transcription RNA has a base sequence complementary to the sequences of DNA

THE FLOW OF GENETIC INFORMATION

2 DNA 1 DNA 1. REPLICATION 2. TRANSCRIPTION 3. TRANSLATION (DNA SYNTHESIS) (RNA SYNTHESIS) (PROTEIN SYNTHESIS) RNA 3 PROTEIN

Transcription and Translation in Cells

Prokaryotic Cell
No nucleus to separate the processes of transcription and translation, when bacterial genes are transcribed, their transcripts can immediately be translated.

Eukaryotic Cell
Transcription occurs in the nucleus to produce a pre-mRNA molecule. The pre-mRNA is typically processed to produce the mature mRNA. mRNA exits the nucleus and is translated in the cytoplasm.

TRANSCRIPTION
DNA RNA

The Central Dogma

Information on Genes flows into proteins The two-step process, transcription and translation, DNA RNA protein.

Onehalf of the DNA strand is used in transcription at a time

5-GCGGACUUACAGAGAUUCCGGAGCCGACAA GCUAGCAAAAAA-3 mRNA 3-CGCCTGAAT GTCTCTAAGGCC TCGGCTGT T CGATCG-5 Template strand of DNA 5-GCGGACTTACAGAGATTC CGGAGCCGACAA GCTAGC-3 Coding template of DNA

Ingredients
rNTPs (Nucleotide tri-phosphates) DNA Template Enzymes

Transcription Unit
Stretch of DNA that codes for an RNA molecule and the sequences necessary for transcription

Protein coding Gene has three critical regions Promoter RNA-coding sequence for the protein Terminator. The promoter is a base-pair sequence: where transcription begins. RNA-coding sequence is a base-pair sequence: coding information for the polypeptide chain specified by the gene. The terminator is a sequence: the end of the mRNA transcript.

Transcription of an RNA Molecule

Transcription begins at the promoter, proceeds through the coding region, and ends at the terminator.

Transcription by RNA Polymerase

RNA polymerase searches for Promoter, unwinds DNA and catalyzes a chemical reaction on a DNA template strand.

Three major types of RNAs

There are 3 types of RNA, each encoded by its own type of gene.

mRNA - Messenger RNA:

typically about 3-500 bases long encode proteins multiple types, usually not abundant, unstable

tRNA - Transfer RNA:

very small - about 100 bases long key role in translation, brings amino acids to ribosomes

rRNA - Ribosomal RNA:

18S, 28S and 5S rRNA structural and functional components of ribosomes highly abundant and stable

mRNA

Eukaryotic mRNAs have a distinct structure at the 5 end

tRNA

Different Types of RNA Polymerase

In Bacteria (simple system)
- all three classes are transcribed by the same RNA polymerase
(RNAP)

In Eukaryotes (complex system)

- each class is transcribed by a different RNA Polymerase RNAP I - rRNAs RNAP II - mRNAs RNAP III - tRNAs & small ribosomal RNAs

Bacterial (Prokaryotic) Transcription

Promoters
- DNA sequences that guide RNAP to the beginning of a gene (transcription initiation site).

Terminators
- DNA sequences that specify then termination of RNA synthesis and release of RNAP from the DNA.

RNA Polymerase (RNAP)

- Enzyme for synthesis of RNA.

Reaction (ordered series of steps)

1) Initiation. 2) Elongation. 3) Termination.

mRNA
Prokaryotes
The sequence of a prokaryotic protein-coding gene is colinear with the translated mRNA; that is, the transcript of the gene is the molecule that is translated into the polypeptide.

Eukaryotes

mRNA

The sequence of a eukaryotic protein-coding gene is typically not colinear with the translated mRNA; Transcript of the gene is a molecule must be processed to remove extra sequences (introns) before it is translated into the polypeptide.
Most eukaryotic protein-coding genes contain segments called introns, which break up the amino acid coding sequence into segments called exons. The transcript of these genes is the pre-mRNA (precursor-mRNA). The pre-mRNA is processed in the nucleus to remove the introns and splice the exons together into a translatable mRNA. That mRNA exits the nucleus and is translated in the cytoplasm.

Modification of pre-mRNA
Eukaryotic mRNAs are modified at both ends during transcription by the addition of a cap to the 5' end and a poly(A) tail to the 3' end. At the 5' end, a cap consists of a modified GTP (guanosine triphosphate). The 5' cap is used as a recognition signal for ribosomes to bind to the mRNA. At the 3' end, a poly(A) tail of 150 or more adenine nucleotides is added. The tail plays a role in the stability of the mRNA.

Processing of pre-mRNA
Splicing
Eukaryotic pre-mRNAs typically include introns. Introns are removed by RNA processing in which the intron is looped out and cut away from the exons by snRNPs, and the exons are spliced together to produce the translatable mRNA. snRNPs (small nuclear ribonucleoprotein particles, complexes of snRNAs and proteins) bind to form the spliceosome, the intron loops out. The intron is excised, and the exons are then spliced together. The resulting mature mRNA may then exit the nucleus and be translated in the cytoplasm.

Some nomenclature conventions

(antisense strand) (noncoding strand)

RNAP
(sense strand) (coding strand)

Other important nomenclature conventions

Transcription Initiation Site Upstream
5 3

Downstream -5 -4 -3 -2 -1 +1 + 2 + 3 + 4 + 5 + 6

3 5

Direction of transcription Template strand

There is no zero

The Central Dogma

Two-step process, transcription and translation, by which the information in genes flows into proteins: DNA RNA Protein.

Translation of the Genetic Code Protein Synthesis

Protein Structure: Amino Acid Building Blocks

The basic building block of a protein is the amino acid. Components of an amino acid

Amino acids are joined together in proteins by peptide bonds.

A peptide bond forms between the carboxyl group of one amino acid (amino acid 1) and the amino group of the adjacent amino acid (amino acid 2).

The Peptide Bond

Translation in a Eukaryotic Cell

mRNA is synthesized in the nucleus and translated on ribosomes in the cytoplasm.

A protein-coding gene is transcribed into a pre-mRNA. Pre-mRNA is processed into a mature mRNA. mRNA exits the nucleus. mRNA is translated on ribosomes to produce the polypeptide chain.

Molecular Components of Translation tRNA, mRNA, Ribosomes

Translation in a Eukaryotic Cell

tRNA (Transfer RNA)
tRNAs bring amino acids to the ribosomes during translation to be assembled into polypeptide chains. tRNAs are encoded by tRNA genes. All tRNA molecules are similar in size and shape. All tRNAs have CCA at the 3' end to which the amino acid attaches. At the other "end" of the tRNA molecule is the anticodon, which, during translation, "reads" the matching codon on the mRNA.

How amino acids are attached to tRNA ?

An enzyme called aminoacyl-tRNA synthetase adds the correct amino acid to its tRNA

Adding an amino acid to tRNA

mRNA (Messenger RNA)

Messenger RNA consists of leader, reading frame and trailer sequences

mRNAs vary in length. Sequences of mRNAs vary because amino acid coding sequences (reading frames) differ, and because leader and trailer sequences differ.

Ribosomes
Organelles on which the mRNA is translated. Consist of two subunits. Each subunit contains rRNA and ribosomal proteins. mRNA binding groove to hold onto the message In translation, the mRNA passes through the ribosome, where the codons are recognized by tRNAs carrying the specified amino acids. Each ribosomal subunit consists of rRNA (ribosomal RNA, encoded by rRNA genes) and ribosomal proteins. The large subunit is 60S: contains 28S, 5.8S, 5S rRNAs and about 50 ribosomal proteins. The small subunit is 40S: contains 18S rRNA and about 30 proteins. S: How fast it sediments during centrifugation

Initiation of Translation

Elongation of Translation

Elongation of Translation

Termination of Translation

Polysomes
Several ribosomes can translate an mRNA at the same time, forming what is called a polysome.

Prokaryotic

Eukaryotic

Initiation

Elongation

Termination

 
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