During transcription , a DNA sequence isread by RNA polymerase which produces acomplementary , antiparallel RNA strand.
As opposed to DNA replication, transcription results in an RNAcompliment that includesuracil(U) in all instances wherethymine(T) would have occurredin a DNA compliment.
Transcrpition is the first step leading to geneexpression. The stretch of DNA transcribed into an RNA moleculeis called a transcription unit and encodes at leastone gene. If the gene transcribed encodes for a protein , theresult of transcription is messenger RNA (mRNA), which will then be used to create that protein via theprocess of translation
Alternatively , the transcribed genemay encode for either ribosomalRNA(rRNA) or transfer RNA (tRNA) , othercomponents of the protein–assemblyprocess or other ribozymes.
A DNA transcription unit encoding for aprotein contains not only the sequence thatwill eventually be directly translated into theprotein (the coding sequence) but alsoregulatory sequences that direct and regulatethe synthesis of that protein.
As in DNA replication , DNA is read from 3→5’during transcription. Meanwhile , thecomplementary RNA is created from the 5→3‘direction. •Only one of the two DNA strands, called thetemplate strand,is used for transcription. •The other DNA strand is called the codingstrand, because its sequence is the same asthe newly created RNA transcript (except forthe substitution of uracil for thymine).
•Transcription is divided into 3 stages:1.initiation2.elongation3.termination.
Initiation In bacteria, transcription begins with thebinding of RNA polymerase to the promoterin DNA. RNA polymerase is a core enzyme consistingof five subunits: 2αsubunits,1βsubunit,1βsubunit,and1ωsubunit At the start of initiation, the core enzyme isassociated with a sigma factor that aids infinding the appropriate – 35 and-10 basepairs downstream of promoter sequences.
Elongation One strand of DNA, the template strand (or noncoding strand) ,isused as a template for RNAsynthesis. As transcription proceeds, RNA polymerasetraverses the template strand and uses base pairingcomplementarity with the DNA template to create anRNA copy. Although RNA polymerase traverses the templatestrand from 3→5, the coding (non- template)strand and newly-formed RNA can also be used asreference points, so transcription can be describedas occurring 5→3.
Elongation This produces an RNA molecule from 5→3, an exactcopy of the coding strand(except that thymines arereplaced with uracils , and the nucleotides arecomposed of a ribose (5- carbon) sugar where DNAhas deoxyribose (one less oxygen atom)in its sugar-phosphate backbone). Unlike DNA replication, mRNA transcription caninvolve multiple RNA polymerases on asingle DNA template and multiple rounds oftranscription (amplification of particular mRNA), somany mRNA molecules can be rapidly producedfrom a single copy of a gene
Elongation Elongation also involves a proofreadingmechanism that can replace incorrectlyincorporated bases.
Termination Bacteria use two differentstrategies for transcriptiontermination:Rho-independent andRho-dependent
TERMINATION In Rho-independent transcription terminationRNA transcription stops when the newlysynthesized RNA molecule forms a G-Crichhairpin loop followed by a run of Us , whichmakes it detach from the DNA template.
Termination In the "Rho-dependent“ type of termination , aprotein factor called "Rho“ destabilizes theinteraction between the template and themRNA, thus releasing the newly synthesized mRNAfrom the elongation complex.
Properties of genetic code The code is universal. All prokaryotic andeukaryotic organisms use the same codon tospecify each amino acid. The code is triplet. Three nucleotides make onecodon. 61 of them code for amino acids and 3viz., UAA, UAG and UGA are nonsense codonsor chain termination codons. The code is degenerate. For a particular aminoacid more than one word can be used.
Properties of genetic code The code is non overlapping. A base in mRNAis not used for two different codons. The code is commaless. There is no specialsignal or commas between codons. The code is non ambiguous. A particular codonwill always code for the same aminoacid, wherever it is found.
Important Codon Facts AUG is the start codon on all viable mRNAmolecules. It also codes for the amino acidMethionine UAA, UAG, UGA are stop codes. Many codons are degenerate --more than onecodon codes for an AA. 43 different combinations give 64 differentcodons
Transfer RNA Transfer RNA (tRNA) is a carriermolecule which picks up amino acidsfrom the cytoplasm and carries themto the mRNA / ribosome complex.AAMethionineU A Canticodon
Ribosomes Bacterial ribosomes consists of two subunitsof unequal size, the larger having asedimentation coefficient of 50 S and thesmaller of 30S. The two ribosomal subunits have irregularshapes which fit together in such a way that acleft is formed through which mRNA passes asthe ribosome moves along it during thetranslation process and from which the newlyformed polypeptide chain emerges.
Circular mRNA Visualized Circular mRNA Visualized byAtomic Force Microscopyby Atomic Force MicroscopyCircular mRNA Visualized by Atomic Force MicroscopyCircular mRNA visualized by Atomicforce Microscopy, pink color showsribosomes
Ribosomal structure-the three tRNA binding sites are:1. A site=holds tRNA that is carrying the nextamino acid to be added2. P site= holds tRNA that is carrying thegrowing polypeptide chain3. E site= where discharged tRNAs leave theribosome
Electron Microscope View• mRNA (purple) binds to a ribosome.• Beads of AA’s (yellow) are joined toform a polypeptide.
Definition: Process of converting informationstored in nucleic acid sequences intoproteins. Sequences of mRNA (messengerRNA)are translated into unique sequence ofamino acids in polypeptidechain(linear order is preservedthroughout !)
Translation is the first stage of proteinbiosynthesis (part of the overall process ofgene expression). Translation is the production of proteins bydecoding mRNA produced in transcription. It occurs in the cytoplasm where theribosomes are located. Ribosomes are made of a small and largesubunit which surrounds the mRNA.
In translation, messenger RNA (mRNA) is decoded toproduce a specific polypeptide according to the rulesspecified by the genetic code. This uses an mRNA sequence as a template to guidethe synthesis of a chain of amino acid that form aprotein. Many types of transcribed RNA , such as transferRNA, ribosomal RNA, and small nuclear RNA are notnecessarily translated into an amino acid sequence.
Translation Phases Translation proceeds in four phases: –activation, –initiation, –elongation and –termination(all describing the growth of the amino acid chain, orpolypeptide that is the product of translation). Amino acids are brought to ribosomes and assembledinto proteins.
Activation In activation, the correct amino acid iscovalently bonded to the correct transfer RNA(tRNA). While this is not technically a step intranslation, it is required for translation toproceed. The amino acid is joined by its carboxyl groupto the 3‘ OH of the tRNA by an ester bond. When the tRNA has an amino acid linked to it, itis termed "charged".
Initiation In Prokaryotes initiation requires the largeand small ribosome subunits, the mRNA, theinitiator tRNA and three initiation factors(IF1,IF2,IF3) and GTP. The overall sequence ofthe event is as follows: IF3 bind to the free 30S subunit, this helps toprevent the large subunit binding to it with outan mRNA molecule and forming an inactiveribosome. IF2 complexed with GTP and IF1 then binds tothe small subunit. It will assist the chargedinitiator tRNA to bind.
Initiation The 30S subunit attached to an mRNA moleculemaking use of the ribosome binding site (RBS) onthem RNA. The initiator tRNA can then bind to the complexby base pairing of its anticodon with the AUGcodon on mRNA. At this point, IF3 can be released, as its role inkeeping the subunits a part and helping themRNA to bind are complete. This complex is called 30S initiation complex.
Initiation The 50S subunit can now bind, whichdisplace IF1 and IF2, and the GTP ishydrolysed in this energy consumingstep. This complex is called as 70S initiationcomplex.
The assembled ribosome has two tRNAbinding sites. These are called the A and P sites, foraminoacyl and peptidylsites. The A site is where incoming aminoacyl tRNAmolecules bind, and the Psite is where thegrowing polypeptide chain is usually found. These sites are in the cleft of small subunitand contain adjacent codons that are beingtranslated.
Elongation StepsElongation is divided into three steps:1. Aminoacyl tRNA delivery EF-Tu is required to deliver the aminoacyltRNA to the A site and energy is consumed inthis step by the hydrolysis of GTP. The released EF-Tu. GDP complex isregenerated with the help of EF-Ts. In the EF-Tu EF-Ts exchange cycle EF-Tsdisplaces the GDP and subsequently isdisplaced itself by GTP.
Elongation StepsThe resultant EF-Tu.GTP complexis now able to bind anotheraminoacyl tRNA and deliver it tothe ribosome.All aminoacyl tRNAs can form thiscomplex with EF-Tu except theinitiator tRNA.
Elongation Steps2. Peptide bond formation After aminoacyl-tRNA delivery, the A- and P-sites are both occupied and the two aminoacids that are to be joined are in closeproximity. The peptidyl transferase activity of the 50Ssubunit can now form a peptide bond betweenthese two amino acids without the input of anymore energy, since energy in the form of ATPwas used to charge the tRNA.
Peptide chain formationA U G C G G U U U A G G C C C U A GA U CstopAA1 AA4AA3AA2 AA5
Elongation Steps3.Translocation. A complex of EF-G (translocase) and GTPbinds to the ribosome and, in an energyconsuming step, the discharged tRNA isejected from the P-site, the peptidyl-tRNA ismoved from the A-site to the P-site and themRNA moves by one codon relative to onecodon to the ribosome.
Elongation Steps GDP and EF-G are released, the latterbeing reusable. A new codon is nowpresent in the vacant A-site. The cycle is repeated until one of thetermination codons (UAA, UAG and UGA)appear in the A-site.
Termination Termination of the polypeptide happens whenthe Asite of the ribosome faces a stop codon(UAA, UAG, or UGA). When this happens, no tRNA can recognizeit, but a releasing factor can recognizenonsense codons and causes the release ofthe polypeptide chain. The 5’end of the mRNA gives rise to theproteins N-terminus, and the direction oftranslation can therefore be stated as N->C.
methionine glycine serine isoleucine glycine alaninestopcodonproteinA U G G G C U C C A U C G G C G C A U A AmRNAstartcodoncodon 2 codon 3 codon 4 codon 5 codon 6 codon 7codon 1
Termination Termination of polypeptide synthesis is signalled by oneof the three termination codons in them RNA (UAA, UAGand UGA) immediately following the last amino acidcodon. In prokaryotes, once a termination codon occupies theribosomal A-site, three termination or releasefactors, viz., the protein RF1, RF2 and RF3 contribute to: The hydrolysis of the terminal peptidyl-tRNA bond. Release of the free polypeptide and the last unchargedtRNA from the P-site The dissociation of the 70S ribosome into its 30S and50S subunits
Termination RF1 recognizes the termination codonUAG and UAA and RF2 recognize UGAand UAA. Either RF1 or RF2 binds at thetermination codon and inducespeptidyl transferase to transfer thegrowing peptide chain to a watermolecule rather than to another aminoacid.Function of RF3 is not known
Polyribosomes A single strand of mRNA is translatedsimultaneously by many ribosomes, spacedclosely together. Such clusters of ribosomes are calledpolysomes or polyribosomes. The simultaneous translation of a singlemRNA by many ribosomes allow highlyefficient use of them RNA.