GENE EXPRESSION THE FLOW OF GENETIC INFORMATION

Chapter outlineThe genetic code - How triplets of the four nucleotides unambiguously specify 20 amino acids, making it possible to translate information from a nucleotide chain to a sequ

6TH WEEK, BIO-1053

GENE EXPRESSION THE FLOW OF GENETIC INFORMATION

General Genetics-BIO1053

6 th week

Chapter outline

The genetic code

- How triplets of the four nucleotides unambiguously specify

20 amino acids, making it possible to translate information from a nucleotide chain to a sequence of amino acids

Transcription: From DNA to RNA

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Transcription: From DNA to RNA

Translation: From mRNA to Protein

Differences in Gene expression between prokaryotes and

eukaryotes

The effect of mutation on gene expression and gene function

The genetic code

The four nucleotides encode 20 amino acids

The genetic code

Triplet codons of nucleotides represent individual amino acids

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61 codons represent the 20 amino acids, 3 codons signify stop

Evidence that a codon is composed of more than one nucleotide, Yanofsky, 1960s

Different point mutations may affect the same amino acid

• Codons must contain >1 nucleotide

Each point mutation affects only one amino acid

• Each nucleotide is part of only one codon

Evidence that a codon is composed of more than one nucleotide, Yanofsky, 1960s

Studies of frameshift mutations showed that codons

consist of three nucleotides

F Crick and S Brenner (1955)

Proflavin-induced mutations in

bacteriophage T4 rIIB gene

• Intercalates into DNA

• Causes insertions and

deletions

2nd treatment with proflavin can

create a 2nd mutation that

restores wild-type function

(revertant)

• Intragenic suppression

Different sets of T4 rIIB mutations generate either a mutant

or a normal phenotype

Codons must be read in

order from a fixed

starting point

Starting point establishes a

reading frame

Intragenic supression

occurs only when wild-type

reading frame is restored

Codons consist of three nucleotides read in a defined

reading frame

Cracking the code: Discovery of mRNA

1950s, studies in eukaryotic cells

Evidence that protein synthesis takes place in cytoplasm

•Deduced from radioactive tagging of amino acids

•

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•Implies that there must be a molecular intermediate

between genes in the nucleus and protein synthesis in the cytoplasm

Discovery of messenger RNAs (mRNAs), molecules for

transporting genetic information

Synthetic mRNAs and in vitro translation determines

which codons designate which amino acids

1961-Marshall Nirenberg and Heinrich Matthaei created

mRNAs and translated to polypeptides in vitro

PolymononucleotidesLater, Har Gobind Khorana, made polydinucleotides and polytrinucleotides,

polytretranucleotides-> synthesis of polypeptides

Cracking the genetic code with mini-mRNAs

Nirenberg and Leder(1965)

Resolved ambiguities in genetic code

In vitro translation with trinucleotide

synthetic mRNAs and tRNAs charged with a radioactive amino acid

Correlation of polarities in DNA, mRNA, and polypeptide

mARN có chiều 5’-> 3’ tương ứng với chiều đầu N-> C của chuỗi polypheptide

Một sợi ADN khuôn

Một sợi còn lại là trình tự giống ARN

Các condon vô nghĩa gây sự kết thúc chuỗi

polypeptides, bao gồm - UAA, UAG, UGA

The genetic code: summary

The code consists of a triplet codons, each of which specifies

an amino acid

Codons are nonoverlapping

The code includes 3 stop codon: UAA, UGA, UAG that

The genetic code: summary

5’-3’ direction of mRNA corresponds with N-terminus to

C-terminus of polypeptide

Mutation modify message encoded in sequence

- Frameshift mutations change reading frame

- Missense mutations change codon of amino acid to another one

- Nonsense mutations change a codon for an amino acids to a stop codon

Do living cells construct polypeptides according to same

rules as in vitro experiment?

Yanofsky: Single-base substitutions can explain the altered amino acids in trp− and trp+ revertants

Missense mutations are single nucleotide substitutions and

conform to the code

Proflavin treatment generates trp- mutants

Further treatment generatees trp+ revertants

Single base deletion (trp-) and an insertion causes reversion (trp+)

The genetic code is almost, but not quite, universal

All living organisms use same basic genetics code

- Translational systems can use mRNA from another

organism to generate protein

- Comparisons of DNA and protein sequence reveal perfect correspondence between codons and amino acids among all organisms

Exceptional genetic codes found in ciliates and

mitochondria, for example: UAA, UAG code for glutamine instead of stop codon; CUA specifies threonine instead of leucine

Transcription: From DNA to RNA

RNA polymerase catalyzes transcription

Promoters are DNA sequences that provide the signal to RNA polymerase for starting transcription

RNA polymerase adds nucleotides in 5’-to-3’ direction

• Formation of phosphodiester bonds using ribonucleotide

Initiation: The beginning of transcription

RNA polymerase binds to promoter sequence located near beginning

of gene

• Sigma (s) factor binds to RNA polymerase ( holoenzyme )

• Region of DNA is unwound to form open promoter complex

• Phosphodiester bonds formed between first two nucleotides

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Elongation: an RNA copy of the gen

σ factor separates from RNA polymerase ( core enzyme )

Core RNA polymerase loses affinity for promoter, moves in 3’-to-5’ direction on template strand

Within transcription bubble, NTPs added to 3’ end of nascent mRNA

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Termination: the end of transcription

Terminators are RNA sequences that signal the end of

transcription

intrinsic (don’t require additional factors)

• Usually form hairpin loops (intramolecular H-bonding)

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Information flow

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The promoters of 10 different bacterial genes

Most promoters are upstream to the transcription start pointRNA polymerase makes strong contacts at -10 and -35

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In eukaryotes, RNA is processed after transcription

Transcribed bases

Capping enzyme adds a "backward" G to the 1 st nucleotide of a primary transcript

Methyl transferase add methyl group to this G and to one or two of the

nucleotides

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bases

Processing adds a tail to the 3' end of eukaryotic mRNAs

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RNA splicing removes introns

Exons – sequences found in a gene’s DNA and mature mRNA (expressed regions)

Introns – sequences found in DNA but not in mRNA

The human dystrophin gene: An extreme example of RNA

splicing

Splicing removes introns from a primary transcript

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RNA processing splices out introns and joins adjacent exons

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Splicing is catalyzed by the spliceosome

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Alternative splicing can produce two different

mRNAs from the same gene

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Trans-splicing combines exons from different genes

Occurs in C elegans and a few other organisms

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Differences in transcription between

prokaryotes and eukaryotes

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Differences in transcription between

prokaryotes and eukaryotes

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