PROTEIN SYNTHESIS (From Gene to Protein) Chapter 17

OVERVIEW DNA RNA Protein Transcription –Synthesis of mRNA from DNA Translation (change of language) –Synthesis of protein (polypeptide) from mRNA (uses tRNA) Gene – section of DNA that codes for a protein to be made Recall – a protein is a chain of amino acids

Figure 17.2 Overview: the roles of transcription and translation in the flow of genetic information

Figure 17.3 The triplet code

CODONS Three base nucleotides that eventually code for a specific amino acid There are 64 codons Marshall Nirenberg (1961) deciphered first codon –Found UUU codes for phenylalanine

Amino acids abbreviated by first 3 letters of name or designated single letter AUG = methionine (met) or start UAA, UAG, and UGA = stop codons Nearly universal language among all living organisms

Figure 17.4 The dictionary of the genetic code

RNA vs. DNA Ribose instead of deoxyribose Uracil instead of thymine Single stranded instead of double stranded

TRANSCRIPTION Making pre-mRNA

INITIATION Promoter – site where RNA polymerase attaches to DNA and starts transcription Transcription factors – proteins that mediate the binding of RNA polymerase (in eukaryotes – huge role in gene regulation)

TATA box – sequence of nucleotides (TATAAAA) that is part of promoter region and binds to transcription factors RNA polymerase attaches to promoter, helix unwinds, and elongation begins

Figure 17.7 The initiation of transcription at a eukaryotic promoter

ELONGATION RNA polymerase adds complementary nucleotides in 5’ to 3’ direction About 60 nucleotides per second

Figure 17.6 The stages of transcription: initiation, elongation, and termination

TERMINATION Elongation stops at or just following a stop codon Transcription Animation Another interactive animation

Figure 17.6 The stages of transcription: initiation, elongation, and termination

RNA PROCESSING In eukaryotes… The 5’ end of pre-mRNA is capped with guanine Poly(A) tail – several adenine added to 3’ end –Protects end –Signal for future ribosome attachment –Help to get mRNA out of nucleus –Help prevent degradation

Figure 17.8 RNA processing; addition of the 5 cap and poly(A) tail

RNA SPLICING In eukaryotes… Large portions of mRNA do not code for parts of a protein Introns – noncoding segments Exons – coding segments snRNPs (small nuclear ribonucleoproteins) combine with proteins to make spliceosome Spliceosomes cut at ends of introns and rejoins remaining exons together (recognize special sequences) Ribozymes – mRNA that catalyzes its own intron removal (not all enzymes are proteins)

Figure 17.9 RNA processing: RNA splicing

Figure The roles of snRNPs and spliceosomes in mRNA splicing

WHY INTRONS? Split genes can code for different proteins or different regions of same polypeptide Introns increase the cross over frequency between 2 alleles which increases diversity (this will make more sense in meiosis)

Figure Correspondence between exons and protein domains

TRANSLATION mRNA to protein

Figure Translation: the basic concept

tRNA Shorter than mRNA Shaped like an “L” A specific amino acid attaches to 3’ end Loop region contains anticodon

Figure 17.13a The structure of transfer RNA (tRNA)

Figure 17.13b The structure of transfer RNA (tRNA)

Aminoacyl-tRNA synthetases - bind correct amino acid to a tRNA –There are 20 of these synthetases so each amino acid has its own enzyme –Driven by hydrolysis of ATP

Figure An aminoacyl-tRNA synthetase joins a specific amino acid to a tRNA

RIBSOMES Made of ribosomal RNA (rRNA) and protein Two subunits: large and small –Join only when translation occurring Three binding sites for tRNA –E = exit site tRNA leaves ribosome –P = peptidyl-tRNA binding site Holds growing polypeptide –A = Aminoacyl-tRNA binding site Holds next tRNA with next amino acid

Figure The anatomy of a functioning ribosome

Figure Structure of the large ribosomal subunit at the atomic level

INITIATION Small ribosomal subunit binds to mRNA at 5’end. Initiator tRNA (with met) binds to P site (H bonds between anitcodon and codon). Large ribosomal subunit attaches with help of proteins GTP supplies energy.

Figure The initiation of translation

ELONGATION H bonds between codon and anticodon (in A site)connect next tRNA with next amino acid GTP needed. A ribozyme catalyzes peptide bond between first and second amino acid. Peptide attached to second tRNA.

mRNA moves through ribosome so that the first tRNA leaves via E site and second tRNA moves to P site. Then the third tRNA comes in to A site. Movement requires GTP. Process continues like a conveyer belt.

Figure The elongation cycle of translation

Wobble effect – third base of anticodon can pair with noncomplementary base of codon (a U can bind to a A or G)

TERMINATION When a stop codon reaches the A site there is no matching anticodon on a tRNA. Release factor protein binds instead. Polypeptide released by hydrolysis. Ribsome disassembles.

Figure The termination of translation

Misc. Polyribosomes - several ribosomes can translate the same mRNA strand All synthesis of all proteins begins in cytoplasm Signal peptide sends protein to ER Signal peptide is recognized by signal recognition particle (SRP) Proteins are transorted via rough ER and can be modified in Golgi body (ex. removal of first met)

Figure Polyribosomes

Figure The signal mechanism for targeting proteins to the ER

Prokaryote vs. Eukaryote Prokaryotes –No introns or TATA box –No 5’ G cap or poly A tail –Translation begins before mRNA is completely made (remember no nucleus) Eukaryotes –Introns and TATA box –Cap and tail (protection for exiting nucleus) –mRNA must leave nucleus before translation can start

Figure Coupled transcription and translation in bacteria

MUTATIONS Mutation – a change in DNA sequence (occurs during DNA replication) Point Mutations cause: –missense mutations change the amino acid –nonsense mutations change the amino acid to a stop codon –silent mutations do not change the amino acid

Two types of Point Mutations –Base pair substitutions replacement of nucleotide –Insertions and Deletions - additions or losses of one or more nucleotides Frameshift mutation - occurs when number of nucleotides inserted or deleted is not 3 or a multiple of 3 Mutation rate is ~1 nucleotide altered in every 10 10

Figure The molecular basis of sickle-cell disease: a point mutation

Figure Categories and consequences of point mutations: Base-pair insertion or deletion

Figure Categories and consequences of point mutations: Base-pair substitution

MUTAGENS Physical or chemical agents that cause DNA to mutate –X-rays –UV light –Radiation –Most carcinogens

A gene is more than just a protein maker. A gene is a region of DNA whose final product is protein or RNA Types of RNA made include –mRNA, tRNA, rRNA, snRNA, SRP RNA (part of signal recognition particle), snoRNA (small nucleolar RNA helps process pre- rRNA), and siRNA (small interfering RNA) and miRNA (micro RNA) both involved in gene regulation

Figure A summary of transcription and translation in a eukaryotic cell