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Chapter 18. Gene Expression and Protein Synthesis. Introduction. The central dogma of molecular biology information contained in DNA molecules is expressed in the structure of proteins gene expression is the turning on or activation of a gene. Transcription.

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Chapter 18
Chapter 18

Gene Expression and Protein Synthesis


Introduction
Introduction

  • The central dogma of molecular biology

    • information contained in DNA molecules is expressed in the structure of proteins

    • gene expression is the turning on or activation of a gene


Transcription
Transcription

  • Transcription: the process by which RNA is synthesized from a DNA template

    • transcription starts when the DNA double helix begins to unwind near the gene to be transcribed (helicase)

    • only one strand of the DNA is transcribed (template strand)

    • ribonucleotides assemble along the unwound DNA strand in a complementary sequence

    • enzymes called polymerases(poly) catalyze transcription: Pol I for rRNA formation, Pol II for mRNA formation, and Pol III for tRNA formation



Transcription2
Transcription

  • A eukaryotic gene has two parts

    • a structural gene that is transcribed into RNA; the structural gene is made of exons and introns

    • a regulatory gene that controls transcription; the regulatory gene is not transcribed but has control elements, one of which is the promoter

    • a promoter is unique to each gene

    • there is always a sequence of bases on the DNA strand called an initiation signal

    • promoters also contain consensus sequences such as the TATA box in which the two nucleotides T and A are repeated many times


Transcription3
Transcription

  • a TATA box lies approximately 26 base pairs upstream

  • all three RNA polymerases interact with their promoter regions via transcription factors that are binding proteins

  • after initiation, RNA polymerase zips up the complementary bases in a process called elongation

  • elongation is in the 5’ -> 3’ direction

  • at the end of each gene is a termination sequence that tells the enzyme “stop the transcription.”


Transcription4
Transcription

  • Pol II has two different forms

    • at its C-terminal domain, it has Ser and Thr repeats that can be phosphorylated

    • when Pol II starts initiation, it is unphosphorylated

    • upon phosphorylation, it catalyzes elongation

    • after termination of the transcription, it is dephosphorylated by a phosphatase

    • in this manner, Pol II is constantly recycled between its initiation and elongation roles


Transcription5
Transcription

  • The RNA products of transcription are not necessarily functional RNAs

    • they are made functional by post-transcription modification

    • transcribed mRNA is capped at both ends

    • the 5’ end acquires a methylated guanine

    • the 3’ end acquires a polyA tail that may contain from 100 to 200 adenine residues

    • once the two ends are capped, the introns are spliced out

    • tRNA is similarly trimmed, capped, and methylated

    • functional rRNA also undergoes post-transcription methylation


Rna in translation
RNA in Translation

  • mRNA, rRNA, and tRNA all participate in translation

  • protein synthesis takes place on ribosomes

  • a ribosome dissociates into a larger and a smaller body

  • in higher organisms, the larger body is called a 60S ribosome; the smaller body is called a 40S ribosome

  • the 5’ end of the mature mRNA is bonded to the 40S ribosome and this unit then joined to the 60S ribosome

  • together the 40S and 60S ribosomes form a unit on which mRNA is stretched out

  • triplets of bases on mRNA are called codons

  • the 20 amino acids are then brought to the mRNA-ribosome complex, each amino acid by its own particular tRNA


tRNA

  • each tRNA is specific for only one amino acid

  • each cell carries at least 20 specific enzymes (synthetases), each specific for one amino acid

  • each enzyme recognizes only one tRNA

  • the enzyme bonds the activated amino acid to the 3’ terminal -OH group of the tRNA by an ester bond

  • at the opposite end of the tRNA molecule is a codon recognition site

  • the codon recognition site is a sequence of three bases called an anticodon

  • this triplet of bases aligns itself in a complementary fashion to the codon triplet on mRNA


tRNA

  • The three-dimensional structure of a tRNA


The genetic code
The Genetic Code

  • Assignments of triplets is based on several types of experiments

    • one of these used synthetic mRNA

    • if mRNA is polyU, polyPhe is formed; the triplet UUU, therefore, must code for Phe

    • if mRNA is poly ---ACACAC---, poly(Thr-His) is formed; ACA must code for Thr, and CAC for His

  • By 1967, the genetic code was broken



Features of the code
Features of the Code

  • all 64 codons have been assigned

  • 61 code for amino acids

  • 3 (UAA, UAG, and UGA) serve as termination signals

  • AUG codes for Met but also serves as an initiation signal

  • only Trp and Met have one codon each

  • more than one triplet can code for the same amino acid; Leu, Ser, and Arg, for example, are each coded for by six triplets

  • the third base is irrelevant for Leu, Val, Ser, Pro, Thr, Ala, Gly, and Arg


Features of the code1
Features of the Code

  • for the 15 amino acids coded for by 2, 3, or 4 triplets, it is only the third letter of the codon that varies. Gly, for example, is coded for by GGA, GGG, GGC, and GGU

  • the code is almost universal: it the same in viruses, prokaryotes, and eukaryotes; the only exceptions are some codons in mitochondria


Reading the genetic code
Reading the Genetic Code

  • Suppose we want to determine the amino acids coded for in the following section of a mRNA

    5’—CCU —AGC—GGA—CUU—3’

  • According to the genetic code, the amino acids for these codons are:

    CCU = Proline AGC = Serine

    GGA = Glycine CUU = Leucine

  • The mRNA section codes for the amino acid sequence of Pro—Ser—Gly—Leu


Translation
Translation

  • Translation: the process whereby a base sequence of mRNA is used to create a protein

  • There are four major stages in protein synthesis

    • activation

    • initiation

    • elongation

    • termination


Protein synthesis

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Protein Synthesis

  • Molecular components of reactions at four stages of protein synthesis


Amino acid activation
Amino Acid Activation

  • Requires

    • amino acids

    • tRNAs

    • aminoacyl-tRNA synthetases

    • ATP, Mg2+

  • Formation on an aminoacyl-tRNA


Amino acid activation1
Amino Acid Activation

  • This two-stage reaction allows selectivity at two levels

    • the amino acid: the aminoacyl-AMP remains bound to the enzyme and binding of the correct amino acid is verified by an editing site on the tRNA synthetase

    • tRNA: there are specific binding sites on tRNAs that are recognized by aminoacyl-tRNA synthetases

      Recognition by an enzyme (synthetase)of its proper tRNA and amino acid is often referred to the second genetic code.


Chain initiation
Chain Initiation

  • Initiation consists of three stages

    • forming the pre-initiation complex: tRNAfMet attaches to small ribosomal subunit

    • migration to mRNA: the pre-initiation complex binds to mRNA

    • forming the full ribosomal complex: the large ribosomal subunit joins the small subunit. The growing peptide chain will bind to the P site of the ribosome


Elongation
Elongation

  • Binding to the A site: A tRNA, whose anticodon matches the next codon on the mRNA, binds to the vacant A site.

  • Forming the 1st peptide bond: On the A site, the alanine is linked to the fMet in a peptide bond by the enzyme peptidyl transferase. The empty tRNA remains on the P site.

  • Translocation: the whole ribosome moves one codon along the mRNA, while the dipeptide is simultaneously translocated from the A site to the P site. The empty tRNA is moved to the E site. When the cycle repeats the tRNA is ejected from the ribosome.


Elongation cont d
Elongation (cont’d)

  • Forming the 2nd peptide bond: A new tRNA, whose anticodon matches the next mRNA codon, enters the now empty A site. The peptidyl transferase creates a peptide bond with the new amino acid, moving the dipeptide from the P site to the A site and forming a tripeptide.

  • These elongation steps are repeated until the last amino acid chain is attached.





Termination
Termination

  • After the final translocation, the next codon reads “stop” (UAA, UAG, or UGA are termination codons). No more amino acids can be added at this point.

  • Releasing factors then cleave the polypeptide chain from the last tRNA. At the end, the whole mRNA is released from the ribosome.


Gene regulation
Gene Regulation

  • Gene regulation: the various methods used by organisms to control which genes will be expressed and when.

    • some regulations operate at the transcriptional level (DNA -> RNA)

    • others operate at the translational level (mRNA -> protein)


Transcriptional level
Transcriptional Level

  • In eukaryotes, transcription is regulated by three elements: promoters, enhancers, and response elements

  • Promoters

    • located adjacent to the transcription site

    • they are defined by an initiator and conserved sequences such as TATA box or GC box

    • different transcription factors bind to different modules of the promoter

    • transcription factors allow the rate of synthesis of mRNA (and from there the target protein) to vary by a factor of up to a million


Promoters
Promoters

  • transcription factors find their targeted sites by twisting their protein chains so that a certain amino acid sequence is present at the surface

  • one such conformational twist is provided by metal-binding fingers (next slide)

  • two other prominent transcription factor conformations are the helix-turn-helix and the leucine zipper

  • transcription factors also possess repressors, which reduce the rate of transcription


Promoters1
Promoters

  • A schematic of a metal-binding finger


Enhancers
Enhancers

  • Enhancers speed up transcription

    • they may be several thousand nucleotides away from the transcription site; a loop in DNA brings the enhancer to the initiation site


Response elements
Response Elements

  • Response elements are activated by their transcription factors in response to an outside stimulus

    • the stimulus may be heat shock, heavy metal toxicity, or a hormonal signal

    • the response element of steroids is in front of and about 250 base pairs upstream from the starting point of transcription


Translational level
Translational Level

  • During translation, there are a number of mechanisms that ensure quality control

  • AARS control

    • each amino acid must attach to the proper tRNA

    • enzymes called aminoacyl-tRNA synthetases (AARS) catalyze this attachment

    • each AARS recognizes its tRNA by specific nucleotide sequences

    • further, the active site of each AARS has two sieving portions (one that prevents larger amino acids from entering, and one that blocks smaller amino acids)


Translational level1
Translational Level

  • Termination control

    • the stop codons must be recognized by release factors

    • the release factor combines with GTP and binds to the ribosomal A site when that site is occupied by the termination codon

  • Post-translational control

    • in most proteins, the Met at the N-terminal end is removed by Met-aminopeptidase

    • certain proteins called chaperones help newly synthesized proteins to fold properly

    • if rescue by chaperones fails, proteasomes may degrade the misfolded protein


Mutations and mutagens
Mutations and Mutagens

  • Mutation: a heritable change in the base sequence of DNA

    • it is estimated that, on average, there is one copying error for every 1010 bases (1 in 10 billion)

    • mutations can occur during replication

    • base errors can also occur during transcription in protein synthesis (a nonheritable error)

    • consider the mRNA codons for Val, which are GUA, GUG, GUC, and GUU

    • if the original codon in DNA is GTA and it is misspelled GTG in the copy, it will be transcribed onto mRNA as GUG which also codes for Val

    • other errors in replication may lead to a change in protein structure and be very harmful


Mutations and mutagens1
Mutations and Mutagens

  • Mutagen: a chemical that causes a base change in DNA (e.g. carcinogens)

  • Many changes in base sequence caused by radiation and mutagens do not become mutations because cells have repair mechanisms called nucleotide excision repair (NER)

    • NER can prevent mutations by cutting out damaged areas and resynthesizing them

  • Not all mutations are harmful

    • certain ones may be beneficial because they enhance the survival rate of the species


Recombinant dna
Recombinant DNA

  • Recombinant DNA: DNA from two sources that have been combined into one molecule

  • One example of the technique begins with plasmids found in the cells of Escherichia coli

    • plasmid: a small, circular, double-stranded DNA molecule of bacterial origin

    • a class of enzymes called restriction endonucleases cleave DNA at specific locations

    • one, for example, may be specific for cleavage of the bond between A-G in the sequence -CTTAAAG-


Recombinant dna1
Recombinant DNA

  • in this example “B ” stands for bacterial gene, and “H for human gene

  • the DNA is now double-stranded with two “sticky ends”, each with free bases that can pair with a complementary section of DNA

  • next, we cut a human gene with the same restriction endonuclease; for example the gene for human insulin


Recombinant dna2
Recombinant DNA

  • the human gene is now spliced into the plasmid by the enzyme DNA ligase

  • splicing takes place at both ends of the human gene and the plasmid is once again circular

  • the modified plasmid is then put back into the bacterial cell where it replicates naturally every time the cell divides

  • these cells now manufacture the human protein, in our example human insulin, by transcription and translation




Protein synthesis1
Protein Synthesis

End

Chapter 18


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