Molecular Biology (MLMB-201)

Molecular Biology

(MLMB-201)

Lecturer:Dr. Mohamed Salah El-Din

Department of Medical Laboratory TechnologyFaculty of Allied Medical Science

Intended Learning Outcomes (ILO’s):

Molecular biology course provides an overview of the molecular basis to cell structure and function.This course focuses on the structure, biosynthesis and function of DNA and RNA on the molecular level and how these interact among themselves and with proteins. Molecular biology techniques are essential for modern biological and medical research. This course will give you an introduction to DNA and RNA standard techniques.

Student will have basic knowledge of:

• Cell organization.• DNA structure and function.• DNA Extraction.• RNA structure and function.• RNA Extraction.• Gene expression and protein biosynthesis.• Agarose gel electrophoresis for DNA/RNA; and SDS-PAGE for protein.• Polymerase Chain Reaction (PCR) – Theory, Types, Application.• Gene library and screening• DNA sequencing

How Are Proteins Made?How Are Proteins Made?(Translation)(Translation)

All Life depends on 3 critical molecules:All Life depends on 3 critical molecules:

DNAsDNAs Hold information on how cell worksHold information on how cell works

RNAsRNAs Act to transfer short pieces of information to different parts of cellAct to transfer short pieces of information to different parts of cell Provide templates to synthesize into proteinProvide templates to synthesize into protein

ProteinsProteins Form enzymes that send signals to other cells and regulate gene Form enzymes that send signals to other cells and regulate gene

activityactivity Form body’s major components (e.g. hair, skin, etc.)Form body’s major components (e.g. hair, skin, etc.)

DNA, RNA, and the Flow of InformationDNA, RNA, and the Flow of Information

TranslationTranscription

Replication

Overview of DNA to RNA to ProteinOverview of DNA to RNA to Protein

A gene is expressed in two stepsA gene is expressed in two steps1)1) Transcription: RNA synthesisTranscription: RNA synthesis2)2) Translation: Protein synthesisTranslation: Protein synthesis

GENE Expression       the Central Dogma of Molecular Biology depicts flow of genetic information          Transcription - copying of DNA sequence into RNA         Translation     -copying of RNA sequence into protein           DNA sequence   RNA sequence   amino acid sequence                TAC                               AUG                                MET triplet sequence in DNA   codon in mRNA         amino acid in protein         Information: triplet sequence in DNA is the genetic word [codon]

Cell Information: Instruction book of LifeCell Information: Instruction book of Life

DNA, RNA, and Proteins DNA, RNA, and Proteins are examples of strings are examples of strings written in either the four-written in either the four-letter nucleotide of DNA letter nucleotide of DNA and RNA (A C G T/U) and RNA (A C G T/U)

or the twenty-letter amino or the twenty-letter amino acid of proteins. Each acid of proteins. Each amino acid is coded by 3 amino acid is coded by 3 nucleotides called codon. nucleotides called codon. (Leu, Arg, Met, etc.)(Leu, Arg, Met, etc.)

Proteins: Workhorses of the CellProteins: Workhorses of the Cell

20 different 20 different amino acidsamino acids different chemical properties cause the protein chains to fold different chemical properties cause the protein chains to fold

up into specific three-dimensional structures that define their up into specific three-dimensional structures that define their particular functions in the cell. particular functions in the cell.

Proteins do all Proteins do all essential workessential work for the cell for the cell build cellular structuresbuild cellular structures digest nutrients digest nutrients execute metabolic functionsexecute metabolic functions Mediate information flow within a cell and among Mediate information flow within a cell and among

cellular communities. cellular communities. Proteins work together with other proteins or nucleic acids Proteins work together with other proteins or nucleic acids

as "molecular machines" as "molecular machines" structures that fit together and function in highly structures that fit together and function in highly

specific, lock-and-key ways. specific, lock-and-key ways.

Terminology for RibosomeTerminology for Ribosome CodonCodon: The sequence of 3 nucleotides in DNA/RNA : The sequence of 3 nucleotides in DNA/RNA

that encodes for a specific amino acid.that encodes for a specific amino acid. mRNA (messenger RNA)mRNA (messenger RNA): A ribonucleic acid whose : A ribonucleic acid whose

sequence is complementary to that of a protein-sequence is complementary to that of a protein-coding gene in DNA.coding gene in DNA.

RibosomeRibosome: The organelle that synthesizes : The organelle that synthesizes polypeptides under the direction of mRNApolypeptides under the direction of mRNA

rRNA (ribosomal RNA)rRNA (ribosomal RNA): The RNA molecules that : The RNA molecules that constitute the bulk of the ribosome and provides constitute the bulk of the ribosome and provides structural scaffolding for the ribosome and catalyzes structural scaffolding for the ribosome and catalyzes peptide bond formation.peptide bond formation.

tRNA (transfer RNA)tRNA (transfer RNA): The small L-shaped RNAs that : The small L-shaped RNAs that deliver specific amino acids to ribosomes according deliver specific amino acids to ribosomes according to the sequence of a bound mRNA.to the sequence of a bound mRNA.

mRNA mRNA Ribosome Ribosome

mRNA leaves the nucleus via nuclear pores.mRNA leaves the nucleus via nuclear pores. Ribosome has 3 binding sites for tRNAs:Ribosome has 3 binding sites for tRNAs:

A-site: position that aminoacyl-tRNA A-site: position that aminoacyl-tRNA molecule binds to vacant sitemolecule binds to vacant site

P-site: site where the new peptide bond is P-site: site where the new peptide bond is formed.formed.

E-site: the exit siteE-site: the exit site Two subunits join together on a mRNA Two subunits join together on a mRNA

molecule near the 5’ end. molecule near the 5’ end. The ribosome will read the codons until AUG The ribosome will read the codons until AUG

is reached and then the initiator tRNA binds is reached and then the initiator tRNA binds to the P-site of the ribosome.to the P-site of the ribosome.

Stop codons have tRNA that recognize a Stop codons have tRNA that recognize a signal to stop translation. Release factors signal to stop translation. Release factors bind to the ribosome which cause the bind to the ribosome which cause the peptidyl transferase to catalyze the addition peptidyl transferase to catalyze the addition of water to free the molecule and releases of water to free the molecule and releases the polypeptide.the polypeptide.

Terminology for tRNA and proteinsTerminology for tRNA and proteins

AnticodonAnticodon: The sequence of 3 nucleotides : The sequence of 3 nucleotides in tRNA that recognizes an mRNA codon in tRNA that recognizes an mRNA codon through complementary base pairing.through complementary base pairing.

C-terminalC-terminal: The end of the protein with the : The end of the protein with the free COOH.free COOH.

N-terminalN-terminal: The end of the protein with the : The end of the protein with the free NH3.free NH3.

Purpose of tRNAPurpose of tRNA

The proper tRNA is chosen by having the corresponding The proper tRNA is chosen by having the corresponding anticodon for the mRNA’s codon. anticodon for the mRNA’s codon.

The tRNA then transfers its aminoacyl group to the The tRNA then transfers its aminoacyl group to the growing peptide chain.growing peptide chain.

For example, the tRNA with the anticodon UAC For example, the tRNA with the anticodon UAC corresponds with the codon AUG and attaches corresponds with the codon AUG and attaches methionine amino acid onto the peptide chain. methionine amino acid onto the peptide chain.

Translation: tRNATranslation: tRNA

Carboxyl end of the protein is released from the tRNA at the Psite Carboxyl end of the protein is released from the tRNA at the Psite and joined to the free amino group from the amino acid attached to and joined to the free amino group from the amino acid attached to the tRNA at the A-site; new peptide bond formed catalyzed by the tRNA at the A-site; new peptide bond formed catalyzed by peptide transferase.peptide transferase.

Conformational changes occur which shift the two tRNAs into the Conformational changes occur which shift the two tRNAs into the E-site and the P-site from the P-site and A-site respectively. The E-site and the P-site from the P-site and A-site respectively. The mRNA also shifts 3 nucleotides over to reveal the next codon.mRNA also shifts 3 nucleotides over to reveal the next codon.

The tRNA in the E-site is releasedThe tRNA in the E-site is released GTP hydrolysis provides the energy to drive this reaction.GTP hydrolysis provides the energy to drive this reaction.

mRNA is translated in 5’ to 3’ direction and the from N-terminal to C-terminus of the polypeptide. Elongation process (assuming polypeptide already began):

tRNA with the next amino acid in the chain binds to the A-site by forming base pairs with the codon from mRNA

Translation: tRNATranslation: tRNA

Terminology for Protein Terminology for Protein FoldingFolding Endoplasmic ReticulumEndoplasmic Reticulum: Membraneous : Membraneous

organelle in eukaryotic cells where lipid organelle in eukaryotic cells where lipid synthesis and some posttranslational synthesis and some posttranslational modification occurs.modification occurs.

MitochondriaMitochondria: Eukaryotic organelle where : Eukaryotic organelle where citric acid cycle, fatty acid oxidation, and citric acid cycle, fatty acid oxidation, and oxidative phosphorylation occur.oxidative phosphorylation occur.

Molecular chaperoneMolecular chaperone: Protein that binds to : Protein that binds to unfolded or misfolded proteins to refold the unfolded or misfolded proteins to refold the proteins in the quaternary structure.proteins in the quaternary structure.

Uncovering the codeUncovering the code

Scientists conjectured that proteins came from Scientists conjectured that proteins came from DNA; but how did DNA code for proteins?DNA; but how did DNA code for proteins?

If one nucleotide codes for one amino acid, then If one nucleotide codes for one amino acid, then there’d be 4there’d be 411 amino acids amino acids

However, there are 20 amino acids, so at least 3 However, there are 20 amino acids, so at least 3 bases codes for one amino acid, since 4bases codes for one amino acid, since 422 = 16 = 16 and 4and 433 = 64 = 64 This triplet of bases is called a “codon”This triplet of bases is called a “codon” 64 different codons and only 20 amino acids means 64 different codons and only 20 amino acids means

that the coding is degenerate: more than one codon that the coding is degenerate: more than one codon sequence code for the same amino acidsequence code for the same amino acid

Revisiting the Central DogmaRevisiting the Central Dogma

In going from DNA to proteins, In going from DNA to proteins, there is an intermediate step there is an intermediate step where mRNA is made from where mRNA is made from DNA, which then makes proteinDNA, which then makes protein This known as This known as The Central The Central

DogmaDogma Why the intermediate step?Why the intermediate step?

DNA is kept in the nucleus, DNA is kept in the nucleus, while protein sythesis happens while protein sythesis happens in the cytoplasm, with the help in the cytoplasm, with the help of ribosomesof ribosomes

The Central Dogma (cont’d)The Central Dogma (cont’d)

RNA RNA Protein: Translation Protein: Translation

Ribosomes and Ribosomes and transfer-RNAstransfer-RNAs (tRNA) run along (tRNA) run along the length of the newly synthesized mRNA, the length of the newly synthesized mRNA, decoding one codon at a time to build a growing decoding one codon at a time to build a growing chain of amino acids (“peptide”)chain of amino acids (“peptide”) The tRNAs have anti-codons, which complimentarily The tRNAs have anti-codons, which complimentarily

match the codons of mRNA to know what protein gets match the codons of mRNA to know what protein gets added nextadded next

But first, in eukaryotes, a phenomenon called But first, in eukaryotes, a phenomenon called splicing occurssplicing occurs Introns are non-protein coding regions of the mRNA; Introns are non-protein coding regions of the mRNA;

exons are the coding regionsexons are the coding regions Introns are removed from the mRNA during splicing so Introns are removed from the mRNA during splicing so

that a functional, valid protein can formthat a functional, valid protein can form

TranslationTranslation

The process of going The process of going from RNA to polypeptide.from RNA to polypeptide.

Three base pairs of RNA Three base pairs of RNA (called a codon) (called a codon) correspond to one amino correspond to one amino acid based on a fixed acid based on a fixed table. table.

Always starts with Always starts with Methionine and ends with Methionine and ends with a stop codona stop codon

Translation, continuedTranslation, continued

Catalyzed by RibosomeCatalyzed by Ribosome Using two different sites, Using two different sites,

the Ribosome continually the Ribosome continually binds tRNA, joins the binds tRNA, joins the amino acids together and amino acids together and moves to the next location moves to the next location along the mRNAalong the mRNA

~10 codons/second, but ~10 codons/second, but multiple translations can multiple translations can occur simultaneouslyoccur simultaneously

Protein Biosynthesis: SummaryProtein Biosynthesis: Summary

There are twenty amino There are twenty amino acids, each coded by acids, each coded by three- base-sequences in three- base-sequences in DNA, called “codons”DNA, called “codons” This code is degenerateThis code is degenerate

The The central dogmacentral dogma describes how proteins describes how proteins derive from DNAderive from DNA DNADNA mRNAmRNA

(splicing?) (splicing?) proteinprotein The protein adopts a 3D The protein adopts a 3D

structure specific to it’s structure specific to it’s amino acid arrangement amino acid arrangement and functionand function

Genetic Code(Amino acid table)

ProteinsProteins

Complex organic molecules made up of amino acid Complex organic molecules made up of amino acid subunitssubunits

2020** different kinds of amino acids. Each has a 1 and 3 different kinds of amino acids. Each has a 1 and 3 letter abbreviation.letter abbreviation.

http://www.indstate.edu/thcme/mwking/amino-acids.html for complete list of chemical structures and for complete list of chemical structures and abbreviations.abbreviations.

Proteins are often enzymes that catalyze reactions.Proteins are often enzymes that catalyze reactions. Also called “poly-peptides”Also called “poly-peptides”

*Some other amino acids exist but not in humans.

Polypeptide v. ProteinPolypeptide v. Protein

A protein is a polypeptide, however to understand the A protein is a polypeptide, however to understand the function of a protein given only the polypeptide sequence function of a protein given only the polypeptide sequence is a very difficult problem. is a very difficult problem.

Protein folding an open problem. The 3D structure Protein folding an open problem. The 3D structure depends on many variables.depends on many variables.

Current approaches often work by looking at the Current approaches often work by looking at the structure of homologous (similar) proteins. structure of homologous (similar) proteins.

Improper folding of a protein is believed to be the cause Improper folding of a protein is believed to be the cause of mad cow disease.of mad cow disease.

http://www.sanger.ac.uk/Users/sgj/thesis/node2.html for more information on folding

Protein FoldingProtein Folding Proteins tend to fold into the lowest Proteins tend to fold into the lowest

free energy conformation.free energy conformation. Proteins begin to fold while the Proteins begin to fold while the

peptide is still being translated.peptide is still being translated. Proteins bury most of its Proteins bury most of its

hydrophobic residues in an interior hydrophobic residues in an interior core to form an core to form an αα helix. helix.

Most proteins take the form of Most proteins take the form of secondary structures secondary structures αα helices and helices and ββ sheets. sheets.

Molecular chaperones, hsp60 and Molecular chaperones, hsp60 and hsp 70, work with other proteins to hsp 70, work with other proteins to help fold newly synthesized help fold newly synthesized proteins.proteins.

Much of the protein modifications Much of the protein modifications and folding occurs in the and folding occurs in the endoplasmic reticulum and endoplasmic reticulum and mitochondria.mitochondria.

Protein FoldingProtein Folding

Proteins are not linear structures, though they Proteins are not linear structures, though they are built that wayare built that way

The amino acids have very different chemical The amino acids have very different chemical properties; they interact with each other after the properties; they interact with each other after the protein is builtprotein is built This causes the protein to start fold and adopting it’s This causes the protein to start fold and adopting it’s

functional structurefunctional structure Proteins may fold in reaction to some ions, and Proteins may fold in reaction to some ions, and

several separate chains of peptides may join together several separate chains of peptides may join together through their hydrophobic and hydrophilic amino acids through their hydrophobic and hydrophilic amino acids to form a polymerto form a polymer

Protein Folding (cont’d)Protein Folding (cont’d)

The structure that a The structure that a protein adopts is vital to protein adopts is vital to it’s chemistryit’s chemistry

Its structure determines Its structure determines which of its amino acids which of its amino acids are exposed carry out the are exposed carry out the protein’s functionprotein’s function

Its structure also Its structure also determines what determines what substrates it can react substrates it can react withwith

Assignment:

As a part of the semester activity, a group of students is selected every week to prepare a short seminar about his/her point of interest in one of the lecture topics. That to be discussed and evaluated during the next lecture.