RNA and Protein Synthesis

How does DNA “code”? DNA inherited by an organism Leads to specific traits by dictating synthesis of proteins DNA directs protein synthesis/gene expression 2 stages - transcription and translation

Central Dogma DNA  RNA  PROTEIN

History of RNA Synthesis George Beadle & Edward Tatum in (‘40s)- ‘One gene, one enzyme’ Function of a gene is to dictate the production of a specific enzyme Beadle 1903-1989 American Nobel Prize 1958 Tatum 1909-1975 American Nobel Prize 1958

After Beadle & Tatum Some genes encode proteins that are not enzymes One gene is responsible for one polypeptide chain, and some proteins have more than one chain One gene, one polypeptide hypothesis

The BIG Picture DNA is transcribed to form RNA RNA is translated to form protein

TRANSLATION TRANSCRIPTION DNA mRNA Ribosome Polypeptide Nuclear envelope TRANSCRIPTION DNA Pre-mRNA RNA PROCESSING mRNA Ribosome Prokaryotic cell - no nucleus, mRNA produced by transcription immediately translated without additional processing TRANSLATION Polypeptide Eukaryotic cell - nucleus provides a separate compartment for transcription. Original RNA transcript (pre-mRNA) processed in various ways before leaving nucleus as mRNA

RNA RNA is used as an intermediary between DNA and proteins RNA is a single strand nucleotide polymer Composition Sugar- Ribose Phosphate group(s) Uracil substitutes for thymine

uracil

Transcription Overview A copy of the DNA is made in the form of mRNA (messenger RNA) in transcription

Translation Overview Translation involves mRNA, tRNA (transfer RNA), and rRNA (ribosomal RNA) coordinating to produce proteins

Translation Overview 2 mRNA has sequences of 3 nucleotides called codons Codons are read in sequences of 3 called triplet code

Translation Overview 3 Codons are written 5’ to 3’ fashion Each codon codes for one amino acid Codons do not overlap

Translation Overview 4 Four bases can combine in 43 combinations– more than enough to code for the 20 naturally occurring amino acids 43 = 64 Why don’t we have 64 amino acids?

Universal Code Genetic code (AA code) nearly universal From simplest bacteria to complex animals Genes can be transcribed & translated after being transplanted from one species to another Tobacco plant with firefly gene

Translation Overview - tRNA tRNA molecule has a sequence of 3 nucleotides- the anticodon Anticodons base pair with the codon in a complementary way Anticodons are written in 3’ to 5’ direction

Translation Overview 6 The E, P, and A are rRNA Ribosomes are composed of proteins and rRNA The E, P, and A are rRNA Ribosome

Transcription: Initiation Synthesis of RNA from the DNA template Main enzyme is RNA polymerase Transcription does not involve a primer - it begins at a promoter site The promoter is a “start” sequence

Transcription: Elongation RNA synthesis proceeds in a 5’-3’ direction copying DNA from the 3’-5’ Upstream- towards 5’ end of mRNA sequence Downstream- towards the 3’ end of mRNA sequence

Transcription: Elongation RNA polymerase moves along the DNA Untwists the double helix, exposing about 10 to 20 DNA bases at a time for pairing with RNA nucleotides

Transcription: Elongation Bacterial promoters are about 40 bases long and are located in the DNA just upstream from the starting point

Transcription: Termination Sequences at the end of the gene act as stop signals Typically only one strand of DNA is transcribed and is called the template strand

Termination (eukaryotes) Enzymes in nucleus modify pre-mRNA (before genetic messages sent to the cytoplasm) mRNA contains additional base sequences that do not directly code for proteins 5’ end: modified nucleotide cap 3’ end: poly-A tail

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5’ Cap & Poly A Tail 5’ Cap Poly A Tail Protect mRNA from hydrolytic enzymes Functions as an “attach here” signal for ribosomes Poly A Tail 50 – 250 nucleotides Same function as 5’ cap Faciliates export from nucleus

RNA Modification RNA splicing Removes introns and joins exons Introns – noncoding regions of DNA Exons – coding portions of DNA Introns stay “in” the nucleus, exons “exit” the nucleus

Spliceosome Splicing accomplished by a spliceosome Consists of a variety of proteins and several small nuclear ribonucleoproteins (snRNPs) Each snRNP has several protein molecules and a small nuclear RNA molecule (snRNA) Each is about 150 nucleotides long

Translation 1 During translation, the nucleic acid message is decoded An amino acid is attached to tRNA before becoming incorporated into a polypeptide To form a polypeptide chain, the amino and carboxyl groups of amino acids are joined

Translation 2 The specific sequence of the amino acids (primary structure) is dictated by the sequence of codons of the mRNA

Translation 3 tRNA is linked to amino acids by aminoacyl-tRNA synthetases This is an energy requiring process

RNA molecules - tRNA RNA molecules have specialized regions with specific functions tRNA molecules have attachment sites for amino acids

RNA molecules tRNA molecules have anticodons that bind to complementary codons of the mRNA If the mRNA codon is UAC, then what is the anticodon present on the tRNA?

RNA molecules tRNA must be recognized by both the specific aminoacyl-tRNA synthetase and the ribosome

RNA molecules tRNA ~ 70 nucleotides long, some generic sections & some unique sections The nucleotide chain is folded back upon itself to form 3 or more loops with unpaired nucleotides exposed

Ribosomes Components of translational machinery come together at the ribosomes Ribosomes are composed of two subunits

Ribosomes The large subunit has a groove into which the small subunit fits Ribosomes are transcribed from DNA, but do not carry information Function as physical site of translation & as a catalyst

Ribosome The A site of the ribosome is where the aminoacyl-tRNA binds The P site is where the tRNA holding the polypeptide chain is positioned

Translation Steps Inititiation Elongation Termination

Initiation Initiation factors (proteins) move an initiation tRNA onto the small ribosomal subunit The codon for the initiation is AUG, which codes for the amino acid methionine

Initiation 2 Initiation complex binds to ribosome recognition sequences on the mRNA, and aligns anticodon of tRNA with the codon of mRNA

Initiation 3 The large ribosomal subunit then binds, forming the functional ribosome

Elongation 1 Addition of new amino acids Initiator tRNA is bound to P site of the ribosome - A site is unoccupied until the next aminoacyl-tRNA moves in

Elongation 2 Energy for this process comes from GTP Peptide bond formation Amino group of the new amino acid & carboxyl group of “old” amino acid

Elongation 3 Protein synthesis proceeds from the amino end to the carboxyl end The tRNA molecule is released from the P site requiring ribozyme, peptidyl transferase Translocation - movement of the growing polypeptide chain from the A site to P site - energy comes from GTP

Elongation 4 Translation of the mRNA proceeds in a 3’ to a 5’ direction, which is the same as the direction of transcription Translocation ensures

Termination Occurs when the mRNA presents the codons UAA, UGA, or UAG; No complementary tRNA Release factors recognize codons The ribosome dissociates into the two subunits

Translation Tutorial http://telstar.ote.cmu.edu/Hughes/HughesArchive/tutorial/polypeptide/tutorial.swf

Polyribosome A single mRNA can make many copies of a polypeptide simultaneously Multiple ribosomes, polyribosomes, may trail along the same mRNA

Mutations are changes in DNA Point mutations – single bp change Inheritable (if occurs in gametes) Mutations are changes in DNA

Point mutation Base pair substitution Some have little/no impact Replacement of a pair of complementary nucleotides with another nucleotide pair Some have little/no impact Silent mutation AA change Similar Nonessential

Missense mutation AA change

Nonsense mutation AA change to stop codon

Mutations are changes in DNA Frameshift mutations- involve the insertion or deletion of a base They result in an entirely different sequence of amino acids because they change the _______________ Mutations are changes in DNA

Frameshift Mutations

Mutations are changes in DNA Transposons - movable sequences of DNA that may move into another area of DNA May disrupt genes, but may inactivate others Transposons have some similarities to retroviruses Mutations are changes in DNA

Transposons

Barbara McClintock (1902-1992) Nobel Prize 1983 Cornell University Discovered the transposons “jumping genes” Studied maize

Mutations Hot spots - regions of DNA more likely to undergo mutations, and are often regions of repeated nucleotides, causing the polymerases to slip

Mutagens Agents that cause mutations Including ionizing radiation Mutations in somatic cells are not passed on to the next generation Some mutagens are also carcinogens