RNA vs. DNA• RNA contains the sugar ribose; DNAcontains deoxyribose.• RNA contains the base uracil; DNAcontains thymine instead.• RNA is usually single stranded; DNA isusually double stranded.• RNA is short: one gene long at most; DNAis long, containing many genes.
RNA Used in Protein Synthesis• messenger RNA (mRNA). A copy of the gene that isbeing expressed. Groups of 3 bases in mRNA, called“codons” code for each individual amino acid in theprotein made by that gene.– in eukaryotes, the initial RNA copy of the gene is called the“primary transcript”, which is modified to form mRNA.• ribosomal RNA (rRNA). Four different RNA moleculesthat make up part of the structure of the ribosome. Theyperform the actual catalysis of adding an amino acid to agrowing peptide chain.• transfer RNA (tRNA). Small RNA molecules that act asadapters between the codons of messenger RNA andthe amino acids they code for.
After Transcription• In prokaryotes, the RNA copy of a gene is messengerRNA, ready to be translated into protein. In fact,translation starts even before transcription is finished.• In eukaryotes, the primary RNA transcript of a geneneeds further processing before it can be translated.This step is called “RNA processing”. Also, it needs to betransported out of the nucleus into the cytoplasm.• Steps in RNA processing:– 1. Add a cap to the 5’ end (7-methyl G)– 2. Add a poly-A tail to the 3’ end– 3. splice out introns.
Summary of RNA processing• In eukaryotes, RNA polymerase produces a “primary transcript”, an exact RNA copy of the gene.• A cap is put on the 5’ end.• The RNA is terminated and poly-A is added to the 3’ end.• All introns are spliced out.• At this point, the RNA can be called messenger RNA. It is then transported out of the nucleus intothe cytoplasm, where it is translated.
tRNA –pick up their specific amino acids from thecytoplasm
Proteins• Proteins are composed of one or more polypeptides• Polypeptides are linear chains of amino acids• The sequence of amino acids in a polypeptide is knownas its “primary structure”.
Translation• Translation of mRNA into protein is accomplished by theribosome, an RNA/protein hybrid. Ribosomes arecomposed of 2 subunits, large and small.• Ribosomes bind to the translation initiation sequence onthe mRNA, then move down the RNA in a 5’ to 3’direction, creating a new polypeptide. The first aminoacid on the polypeptide has a free amino group, so it iscalled the “N-terminal”. The last amino acid in apolypeptide has a free acid group, so it is called the “C-terminal”.• Each group of 3 nucleotides in the mRNA is a “codon”,which codes for 1 amino acids. Transfer RNA is theadapter between the 3 bases of the codon and thecorresponding amino acid.
Transfer RNA• Transfer RNA molecules are short RNAsthat fold into a characteristic cloverleafpattern. Some of the nucleotides aremodified to become things likepseudouridine and ribothymidine.• Each tRNA has 3 bases that make up theanticodon. These bases pair with the 3bases of the codon on mRNA duringtranslation.• Each tRNA has its corresponding aminoacid attached to the 3’ end. A set ofenzymes, the “aminoacyl tRNAsynthetases”, are used to “charge” thetRNA with the proper amino acid.• Some tRNAs can pair with more than onecodon. The third base of the anticodon iscalled the “wobble position”, and it can formbase pairs with several differentnucleotides.
Initiation of Translation• In eukaryotes, ribosomes bind to the 5’ cap, then movedown the mRNA until they reach the first AUG, thecodon for methionine. Translation starts from this point.Eukaryotic mRNAs code for only a single gene.• Note that translation does not start at the first base of themRNA. There is an untranslated region at the beginningof the mRNA, the 5’ untranslated region (5’ UTR).
More Initiation• The initiation processinvolves first joining themRNA, the initiatormethionine-tRNA, and thesmall ribosomal subunit.Several “initiationfactors”(eIF 1-6)--additional proteins--arealso involved. The largeribosomal subunit thenjoins the complex.
Elongation• The ribosome has 2 sites for tRNAs, called P and A. The initialtRNA with attached amino acid is in the P site.• A new tRNA, corresponding to the next codon on the mRNA, bindsto the A site.• The ribosome catalyzes a transfer of the amino acid from the P siteonto the amino acid at the A site, forming a new peptide bond.• The ribosome then moves down one codon.• The now-empty tRNA at the P site is displaced off the ribosome, andthe tRNA that has the growing peptide chain on it is moved from theA site to the P site.• The process is then repeated:– the tRNA at the P site holds the peptide chain, and a new tRNA binds tothe A site.– the peptide chain is transferred onto the amino acid attached to the Asite tRNA.– the ribosome moves down one codon, displacing the empty P site tRNAand moving the tRNA with the peptide chain from the A site to the Psite.
Termination• Three codons are called “stopcodons”. They code for no aminoacid, and all protein-codingregions end in a stop codon.• When the ribosome reaches astop codon, there is no tRNA thatbinds to it. Instead, proteinscalled “release factors” bind, andcause the ribosome, the mRNA,and the new polypeptide toseparate. The new polypeptide iscompleted.• Note that the mRNA continues onpast the stop codon. Theremaining portion is not translated:it is the 3’ untranslated region (3’UTR).
Post-Translational Modification• New polypeptides usually fold themselves spontaneouslyinto their active conformation• Many proteins have sugars, phosphate groups, fattyacids, and other molecules covalently attached to certainamino acids. Most of this is done in the endoplasmicreticulum.• Many proteins are targeted to specific organelles withinthe cell. Targeting is accomplished through “signalsequences” on the polypeptide. In the case of proteinsthat go into the endoplasmic reticulum, the signalsequence is a group of amino acids at the N terminal ofthe polypeptide, which are removed from the final proteinafter translation.
The Genetic Code• Each group of 3 nucleotides on the mRNA is a codon. Since thereare 4 bases, there are 43 = 64 possible codons, which must codefor 20 different amino acids.• More than one codon is used for most amino acids: the geneticcode is “degenerate”. This means that it is not possible to take aprotein sequence and deduce exactly the base sequence of thegene it came from.• In most cases, the third base of the codon (the wobble base) canbe altered without changing the amino acid.• AUG is used as the start codon. All proteins are initially translatedwith methionine in the first position, although it is often removedafter translation. There are also internal methionines in mostproteins, coded by the same AUG codon.• There are 3 stop codons, also called “nonsense” codons. Proteinsend in a stop codon, which codes for no amino acid.
Protein synthesis1. DNA unwinds2. mRNA copy is made of one of the DNA strands.3. mRNA copy moves out of nucleus into cytoplasm.4. tRNA molecules are activated as their complementary amino acidsare attached to them.5. mRNA copy attaches to the small subunit of the ribosomes incytoplasm. 6 of the bases in the mRNA are exposed in theribosome.6. A tRNA bonds complementarily with the mRNA via its anticodon.7. A second tRNA bonds with the next three bases of the mRNA, theamino acid joins onto the amino acid of the first tRNA via apeptide bond.8. The ribosome moves along. The first tRNA leaves the ribosome.9. A third tRNA brings a third amino acid10. Eventually a stop codon is reached on the mRNA. The newlysynthesised polypeptide leaves the ribosome.
Transcription 1(making a mRNA copy of DNA)•The part of the DNA molecule (the gene) that the cell wants theinformation from to make a protein unwinds to expose the bases.•Free mRNA nucleotides in the nucleus base pair with one strandof the unwound DNA molecule.
Transcription 2•The mRNA copy is made with the help of RNA polymerase. This enzymejoins up the mRNA nucleotides to make a mRNA strand.•This mRNA strand is a complementary copy of the DNA (gene)•The mRNA molecule leaves the nucleus via a nuclear pore into thecytoplasm
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) basepairs 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 atposition A.•The enzyme peptidyl transferase forms a peptide bond between the two aminoacids.•The first tRNA (without its amino acid) leaves the ribosome.
Translation 2The 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 aminoacid.A growing polypeptide is formed in this way until a stop codon is reached.
End of TranslationA stop codon on the mRNA is reached and this signals the ribosome to leavethe mRNA. A newly synthesised protein is now complete!