TRANSLATION

• Translation involves “decoding” a messenger RNA (mRNA)

• The translation using its information to build a polypeptide or chain of amino acids.

• A polypeptide is basically a protein with the technical difference being that some large proteins are made up of several polypeptide chains.

The Genetic Code

• Triple : a sequence of three base (a codon ) is needed to specify one amino acid

• No overlapping code : no base are shared between consecutive codon

• Continuous code

• Degenerate : more than one codon can code for the same amino acid

• Universal : same in all organisms

• In an mRNA, the instructions for building a polypeptide come in groups of three nucleotides called codons.

• Have 64 codon

• There are 61 different codons for amino acids

• One codon, AUG, is a “start” signal to kick off translation it also specifies the amino acid methionine. (anticodon for AUG is UAC)

• Three codon ( UAA , UAG and UGA ) “stop” codons mark the polypeptide as finished. (don’t have anticodon).

Translation Requirement

• mRNA (codon) , G C C

• tRNA (anticodon), C G G

• rRNA (ribosome)

• various proteion factors

Step Of Translation The Genetic Message

Step 1 : amino acid activation

• In translation, the codons of an mRNA are read in order from the 5' end to the 3' end  by molecules called transfer RNAs (tRNA).

• tRNA has an anticodon, a set of three nucleotides that binds to a matching mRNA codon through base pairing.

• tRNAs bind to mRNAs inside of a protein-and-RNA structure (ribosome).

• As tRNAs enter slots in the ribosome and bind to codons, their amino acids are linked to the growing polypeptide chain (Met-Ile-Ser) in a chemical reaction.

• The end result is a polypeptide whose amino acid sequence mirrors the sequence of codons in the mRNA.

• Free energy of hydrolysis of ATP provides energy for bond formation

Step 2 :Chain Initiation

• In  all organisms , synthesis of polypeptide chain starts at the N- terminal end and grows to C- terminus.

• Initiation required: tRNAfmet  ,initiation codon (AUG) of mRNA ,30S ribosomal subunit,50 ribosomal subunit,initiation factors (Ifs) and GTP,Mg2+

• A ribosome (which comes in two pieces, large and small)

• An mRNA with instructions for the protein we'll build

• An "initiator" tRNA carrying the first amino acid in the protein, which is almost always methionine (Met)

• During initiation, these pieces must come together in just the right way .They form the initiation complex.

• Inside our cells and the cells of other eukaryotes, translation initiation , the tRNA carrying methionine attaches to the small ribosomal subunit.

• Together, they bind to the 5' end of the mRNA by recognizing the 5' GTP cap (added during processing in the nucleus).

• Then, they "walk" along the mRNA in the 3' direction, stopping when they reach the start codon (often, but not always, the first AUG).

• Example : tRNAfmet contain the triple 3’-UAG-5’and the triple base pairs with 5’-AUG-3’ in mRNA.

• 3’-UAC-5’ triplet on tRNAfmet recognizes the AUG triplet (the start signal) when it occurs at the beginning of the mRNA sequence that direct polypeptide synthesis (bind at P site).

Eukaryotic translation initiation

                                                       

Bacterial Translation Initiation

• In bacteria, the situation is a little different. Here, the small ribosomal subunit doesn't start at the 5' end of the mRNA and travel toward the 3' end. 

• Instead, it attaches directly to certain sequences in the mRNA. 

• These Shine-Dalgarno sequences come just before start codons and "point them out" to the ribosome.

Step 3: Chain Elongation

• Elongation is when the polypeptide chain gets longer.

• Uses 3 biding sites for tRNA  present on the 50S subunit of the 70S ribosome: P (peptidyl) site, A(aminoacyl) site ,E (exit) site.

• Requires: 70S ribosome, codons of mRNA , aminoacyl-tRNAs, elongation factors (EF),GTP and Mg2+

• First : a fresh codon is exposed in another slot, called the A site.  

                  : The A site will be the "landing site" for the next tRNA, one whose anticodon is a perfect

                     (complementary) match for the exposed codon.

                  :an aminoacyl-tRNA is bound to the A site 

                  : the P site is already occupied by tRNAfmet 

                  : 2nd amino acid bound to 70S initiation complex.Defined by the mRNA.

     Second : the peptide bond is formed ,the P site is uncharged.

     Third    : the uncharged tRNA is released (E site)

                  : the next peptidyl-tRNA is translocated to the P site

                  : the next aminoacyl-tRNA occupies the empty A site

• Translation read the mRNA from 5’ to 3’ direction

• Ribosom moves toward 3-end 

• Polypeptide sequence grows from N-end to C-end

Step 4 : Chain termination

• Termination happens when a stop codon in the mRNA (UAA, UAG, or UGA) enters the A site.Stop codons have no tRNA,(anticodon).

• Stop codons are recognized by proteins called release factors, which fit neatly into the P site (though they aren't tRNAs).

• Release factor (RFs) which either binds to UAA and UAG or UGA.

• GTP which is bound to RF

• The entire complex dissociates setting free the complete polypeptide ,the release factors,tRNA  mRNA ,and the 30S and 50S ribosomal subunits.

TRANSCRIPTION

Transcription is the synthesis of an RNA strand from a DNA template.

• A gene's protein building instructions are transcribed to messenger RNA (mRNA).

• mRNA carries the code from DNA to the ribosomes where translation into a protein occurs.

The Three Steps of Transcription

Step 1 : Initiation

• RNA polymerase binds to DNA at a specific sequence of nucleotides called the promoter.

• The promoter contains an initiation site where transcription of the gene begins.

• RNA polymerase than unwinds DNA at the beginning of the gene.

Step 2 : Elongation

• Only one of the unwound DNA strands acts as a template for the RNA synthesis.

• RNA polymerase can only add nucleotides to the 3' end of the strand so like DNA, RNA must be synthesized in the 5' to 3' direction.

• Free ribonucleosides triphosphates (ATP, CTP,GTP and UTP) from the cytoplasm are paired up with their complementary base on the exposed DNA template.

• RNA polymerase joins the ribonucleoside triphosphates to form an mRNA strand.

• As RNA polymerase advances, the process continues.

• The DNA that has been transcribed, re-winds to form a double helix.

Step 3 : Termination

• RNA polymerase continues to elongate until it reaches the terminator, a specific sequence of nucleotides that signals the end of transcription.

• When transcription stops, mRNA polymerase and the new mRNA transcript are released from DNA.

• The DNA double helix reforms.

• The termination sequence usually consists of a series of adjacent adenines preceded by a nucleotide palindrome.

• This gives an RNA molecule that assumes a stem-and loop configuration.

• This configuration stops RNA polymerase from transcribing any further.

• 2 type of termination mechanisme is Intrinsic termination and Termination involves rho (p) protein.

• Intrinsic termination is controlled by specific sequences .

• Termination involves rho (p) is rho-dependent termination sequences cause hairpin loop to form

Transcription : Initiation, Elongation and Termination Steps

DNA REPLICATION

• DNA replication is the biological process of producing two identical replicas of DNA from on original DNA molecule.

• DNA is made up of a double helix of two complementary strands.

DNA Replication is Semi-Conservative

• DNA replication of one helix of DNA results in two identical helices. If the original DNA helix is called the "parental" DNA, the two resulting helices can be called "daughter" helices.

• DNA creates "daughters" by using the parental strands of DNA as a template or guide. 

• Each newly synthesized strand of DNA (daughter strand) is made by the addition of a nucleotide that is complementary to the parent strand of DNA.

• In this way, DNA replication is semi-conservative, meaning that one parent strand is always passed on to the daughter helix of DNA.

The Semi-Conservative Nature of DNA Replication

DNA Replication Process

The Three Steps of DNA Replication

Step 1 : Initiation

• The first step in DNA replication is the separation of the two DNA strands that make up the helix that is to be copied. 

• DNA Helicase untwists or “unzipped” the helix at locations called replication origins. 

• The replication origin forms a Y shape, and is called a replication fork. 

• The replication fork moves down the DNA strand, usually from an internal location to the strand's end. 

• The result is that every replication fork has a twin replication fork, moving in the opposite direction from that same internal location to the strand's opposite end.

• Single-stranded binding proteins (SSB) work with helicase to keep the parental DNA helix unwound. 

• It works by coating the unwound strands with rigid subunits of SSB that keep the strands from snapping back together in a helix. 

• The SSB subunits coat the single-strands of DNA in a way as not to cover the bases, allowing the DNA to remain available for base-pairing with the newly synthesized daughter strands.

• The cell prepares for the next step, elongation, by creating short sequences of RNA called primers that provide a starting point of elongation.

Replication fork 

Step 2 : Elongation

• Enzyme DNA polymerases are responsible creating the new strand by a process called elongation. 

• Five different known types of DNA polymerases in bacteria and human cells which is polymerase I, II, III, IV and V. 

• Polymerase III is the main replication enzyme, while polymerase I, II, IV and V are responsible for error checking and repair. 

• The enzyme DNA polymerase controls elongation, which can occur only in the leading direction.

• DNA is directional in both strands, signified by a 5' and 3' end. This notation signifies which side group is attached the DNA backbone. 

• The 5' end has a phosphate (P) group attached, while the 3' end has a hydroxyl (OH) group attached. 

• This directionality is important for replication as it only progresses in the 5' to 3' direction.

• However, the replication fork is bi-directional; as in the picture, the leading strand is synthesized continuously in the 5’ to 3’ direction toward the replication fork while lagging strand is synthesized discontinuously (Okazaki fragments) also in the 5’ to 3’ direction, but away from the replication fork. 

DNA Replication : Elongation

Step 3 : Termination

• Once all of the bases are matched up (A with T, C with G), an enzyme called exonuclease strips away the primer(s). 

• Then, these primers are replaced with appropriate bases. Another exonuclease “proofreads” the newly formed DNA to check, remove and replace any errors. 

• Another enzyme called DNA ligase joins Okazaki fragments together forming a single unified strand. 

• The ends of the linear DNA present a problem as DNA polymerase can only add nucleotides in the 5′ to 3′ direction.

• The ends of the parent strands consist of repeated DNA sequences called telomeres. Telomeres act as protective caps at the end of chromosomes to prevent nearby chromosomes from fusing. 

• A special type of DNA polymerase enzyme called telomerase catalyzes the synthesis of telomere sequences at the ends of the DNA. 

• Once completed, the parent strand and its complementary DNA strand coils into the familiar double helix shape. 

• In the end, replication produces two DNA molecules, each with one strand from the parent molecule and one new strand.

DNA Replication : Initiation, Elongation, and Termination Steps

Replication Enzymes

DNA replication would not occur without enzymes that catalyze various steps in the process. Enzymes that participate in the eukaryotic DNA replication process include:

DNA Helicase

• unwinds and separates double stranded DNA as it moves along the DNA. It forms the replication fork by breaking hydrogen bonds between nucleotide pairs in DNA.

DNA Primase

• a type of RNA polymerase that generates RNA primers. Primers are short RNA molecules that act as templates for the starting point of DNA replication.

DNA Polymerases

• synthesize new DNA molecules by adding nucleotides to leading and lagging DNA strands. 

• 5 type of DNA polymerase :

          🍀DNA-Pol I: repair and patching of DNA
          🍀DNA-Pol III: responsible for the polymerization of the newly formed DNA strand
          🍀DNA-Pol II, IV, and V: proofreading and repair enzymes

Topoisomerase or DNA Gyrase

• unwinds and rewinds DNA strands to prevent the DNA from becoming tangled or supercoiled.

DNA Ligase

• joins DNA fragments together by forming phosphodiester bonds between nucleotides.

Exonucleases

• group of enzymes that remove nucleotide bases from the end of a DNA chain.

RNA STRUCTURE

• RNA is typically single stranded and is made of ribonucleotides that are linked by phosphodiester bonds.

• A ribonucleotide in the RNA chain contains ribose (the pentose sugar), one of the four nitrogenous bases (A, U, G, and C), and a phosphate group.

• The basic components of RNA are the same than for  DNA with two major differences. The pyrimidyne base uracil replace thymine and ribose replace deoxyribose. Adenine and Uracil for a base pair formed by two hydrogen bonds.

Classification of RNA

RNA molecules are classified according to their structure and function.

Transfer RNA, tRNA :

• The tRNA molecule has a distinctive folded structure with three hairpin loops that form the shape of a three-leafed clover.

• One of these hairpin loops contains a sequence called the anticodon, which can recognize and decode an mRNA codon

• the tRNA transfers the appropriate amino acid to the end of the growing amino acid chain.

• Then the tRNAs and ribosome continue to decode the mRNA molecule until the entire sequence is translated into a protein.

Ribosomal RNA, rRNA:

• a ribonucleic acid found in ribosomes, the site of protein synthesis

• only a few types of rRNA exist in cells

• in both prokaryotes and eukaryotes, ribosomes consist of two subunits- one larger than the other (eg. 50S and 30S)

Messenger RNA, mRNA:

• a ribonucleic acid that carries coded genetic information from DNA to ribosomes for the synthesis of proteins

• single stranded

• biosynthesis is directed by information encoded on DNA

• a complementary strand of mRNA is synthesized along one strand of an unwound DNA, starting from the 3’ end

DNA STRUCTURE

1° structure

Primary Structure : the sequence of bases along the pentose-phosphodiester backbone of a DNA molecule.

• Base sequence is read from the 5’ end to 3’ end

• System of notation single letter ( A, G, C and T )

2° Structure

Secondary Structure : the ordered arrangement of nucleic acid strand

• The double helix model of DNA 2° structure

• Double helix : two antiparallel poly nucleotide strands are coiled in a right-handed manner about the same axis.

Base Pairing

• Base pairing is complimentary

• A major factor stabilizing the double helix is base pairing by hydrogen bonding between T-A and between C-G

T-A base pair comprised of 2 hydrogen bonds

G-C base pair comprised of 3 hydrogen bonds

3° Structure

Tertiary Structure : the three-dimensional arrangement of all atoms of nucleic acid (supercoiling)

• If the DNA is twisted in the direction of the helix, it is said that the positive supercoiling, and the bases are held together more closely.

• If the DNA is twisted in the opposite direction is called negative supercoiling, and away basis. In nature, most DNA has slight negative supercoiling that is produced by enzymes called topoisomerases.

4° Structure

Quaternary Structure : The structure of chromatin

• Each ‘bead’ is a nucleosome. Nucleosome consist of DNA wrapped around histone core.

• Histone : a protein, found associated with eukaryotic DNA

• Chromatin : DNA molecules wound around particles of histones in a beadlike structure.

NUCLEIC ACID BIOCHEMISTRY

DO YOU KNOW WHAT IS NUCLEIC ACID ?

Nucleic acids are linear polymers that consist of monomers called nucleotides. 

Each nucleotide carries 

- a sugar

- a nitrogenous base

- a phosphate group

There are two types of nucleic acids in a nature 

- deoxyribose nucleic acids (DNA)

- ribonucleic acids (RNA). 

DNA contains the deoxyribose sugar and typically exists in a double-helix form. 

Functions of DNA are :

- to store the genetic information and keep it readily accessible to the cell 

- to pass down genetic information to offspring during reproduction. 

RNA molecules contain the ribose sugar and exist predominately as single-strand molecules.

Functions of RNA are :

- transcribes the genetic information into a form that is easy to understand and read by the cell 

- assist in protein synthesis.