Chapter 15 Genes and How They Work

Bonus Topics: significance of Beadle and Tatums Neurospora experiments how did Crick and Brenner crack the code? how did Nirenberg and Khorana determine what each codon meant?

The central dogma of molecular biology states that information flows in one direction:

DNA
Transcription:

RNA

protein

the flow of information from DNA to RNA

Translation:
the flow of information from RNA to protein

The Genetic Code

Question: how can 4 nucleotides (A, T, G, C) could encode more than 20 amino acids? 1 nt how many amino acids? Answer: only four! 2 nts how many amino acids? AA AT How many aas could result? TA CA 3 nts how many amino acids? hmm.. Lets see, theres TT AC TC CT CC AG TG CG GG GA GT GC

The Genetic Code

codon: set of 3 nts in the mRNA that specifies a particular amino acid

The Genetic Code

codon: set of 3 nucleotides that specifies a particular amino acid start codon: the codon (AUG) used to signify the start of translation stop codons: 3 codons (UUA, UGA, UAG) in the genetic code used
to terminate translation

The remainder of the code is degenerate

meaning that some aas are specified by more than one codon (reverse never true: single codon specifying more than one aa) degeneracy is not uniform (see Table 15.1) the degenerate base is usually at the third position (wobble)

 template strand: strand of the DNA double helix used to make RNA coding strand: strand of DNA that is complementary to the template strand RNA polymerase reads the template strand ( the mRNA is (almost) identical to the coding strand!)

DNA RNA (transcription) 3 stages:

initiation: RNA polymerase identifies where to begin transcription elongation: RNA nucleotides are added to the 3 end of the new RNA termination: RNA polymerase stops transcription when it encounters terminators in the DNA sequence

Multiple types of RNA Gene expression requires the participation of:

messenger RNA (mRNA): carries the information from DNA that encodes proteins ribosomal RNA (rRNA): is a structural component of the ribosome transfer RNA (tRNA): carries amino acids to the ribosome for translation

also
small nuclear RNA (snRNA): involved in processing premRNA signal recognition particle (SRP): composed of protein and RNA and involved in directing mRNA to the RER micro-RNA (miRNA): regulatory role

Prokaryotic Transcription
Prokaryotes contain a single type of RNA polymerase found in 2 forms: core polymerase is capable of RNA elongation but not initiation

holoenzyme is composed of the core enzyme and the sigma factor which is required for transcription initiation

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A transcriptional unit

extends from the promoter to the terminator promoter

a DNA sequence for the binding of RNA polymerase start site (+1): the first base to be transcribed 2 sites important for important for positioning RNA pol (both where on the mRNA and in correct direction)

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Transcription initiation at the promoter

subunit recognizes the promoter sites (TATAAT at -10 and the TTGACA at -35) subunit binds to the DNA and opens the helix at the TATAAT site creating the transciption bubble

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The transcription bubble consists of

RNA polymerase DNA template growing mRNA transcript

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Termination:
transcription stops when the transcription bubble encounters terminator sequences

often consist of series of C-Gs followed by A-Ts followed by 4 or more Us forms a hairpin loop causing the RNA strand to dissociate

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In prokaryotes, transcription and translation are often coupled

Homework Why cant this occur in eukaryotes?

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Eukaryotic Transcription
3 RNA polymerases in eukaryotes each RNA polymerase recognizes its own promoter more in CH16 RNA polymerase I transcribes rRNA RNA polymerase II transcribes mRNA and some snRNA RNA polymerase III transcribes tRNA and some other small RNAs

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Eukaryotic Transcription Complex

initiation of transcription of mRNA requires a series of transcription factors (proteins that act to bind RNA polymerase to the promoter and initiate transcription)

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Post-transcriptional modifications of eukaryotic transcripts addition of a 5 cap (methylated GTP) addition of a 3 poly-A tail removal of non-coding sequences (introns)

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pre-mRNA Splicing introns = non-coding sequences exons = sequences that will be translated

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The spliceosome:
responsible for removing introns and splicing exons together small ribonucleoprotein particles (snRNPs) within the spliceosome recognize the intron-exon boundaries

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RNA protein (translation)

3 stages:
initiation: mRNA, tRNA, and ribosome come together elongation: tRNAs bring amino acids to the ribosome for incorporation into the polypeptide termination: ribosome encounters a stop codon and releases polypeptide

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 tRNA molecules carry amino acids to the ribosome for incorporation into a polypeptide
specific enzymes (see next figure) add amino acids to the acceptor arm of tRNA the anticodon loop contains 3 nucleotides complementary to mRNA codons

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tRNA charging reactions:

aminoacyl-tRNA synthetases add amino acids to the acceptor arm of each tRNA 20 different aminoacyl-tRNA synthetases (unique synthetase for each of the 20 different aas) requires energy (ATP is used)

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The ribosome has multiple tRNA binding sites:

P site: binds the tRNA attached to the growing peptide chain A site: binds the tRNA carrying the next amino acid E site: binds the tRNA that carried the last amino acid

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The ribosome has two primary functions: decode the mRNA form peptide bonds
reading frame: the series of nucleotides read in sets of 3 (codons) only 1 reading frame is correct for encoding the correct sequence of amino acids peptidyl transferase is the enzymatic component of the ribosome which forms peptide bonds between amino acids as the ribosome decodes the mRNA

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Prokaryotes: initiation of translation requires the formation of the initiation complex including
an initiator tRNA charged withN-formylmethionine the small ribosomal subunit mRNA strand the ribosome binding sequence of mRNA is complementary to part of rRNA

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Translation elongation involves the addition of amino acids a charged tRNA binds to the A site if its anticodon is complementary to the codon at the A site peptidyl transferase forms a peptide bond the ribosome moves down the mRNA in a 5 to 3 direction

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Elongation cycle
the ribosome moves (translocates) down the mRNA in a 5 to 3 direction each new charged tRNA is brought to the A site by an elongation factor called EF-Tu (which is also bound to GTP) elongation continues until a stop codon is reached

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Termination
termination codon no corresponding tRNAs release factors breaks the bond holding the polypeptide to the P site tRNA

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Eukaryotes: translation may occur on ribosomes in the cytoplasm or on ribosomes of the RER signal sequences at the beginning of the polypeptide sequence bind to the signal recognition particle (SRP) the signal sequence and SRP are recognized by RER receptor proteins
the signal sequence/SRP holds the ribosome on the RER as the polypeptide is synthesized it passes through a pore into the interior of the RER

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Mutation: altered genes

Point mutations: alter a single base base substitution mutations: substitute one base for another transitions or transversions also called missense mutations nonsense mutations: create stop codon frameshift mutations: caused by insertion or deletion of a single base

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Chromosomal mutations: change the structure of a chromosome

deletions: part of chromosome is lost duplication: part of chromosome is copied inversion: part of chromosome in reverse order translocation: part of chromosome is moved to a new location
Mutations: good or bad? too much genetic change (mutation) can be harmful to the individual however, genetic variation (caused by mutation) is necessary for evolutionary change of the species

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