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Chapter 10: From DNA to Protein: Gene Expression
Not all genes code for polypeptides
Some are transcribed to RNA but not translated to polypeptides; the RNA's have other functions
Molecular Biology: the study of nucleic acids and proteins: often focuses on gene expression
Genes are expressed in two steps:
Transcription: information from a DNA sequence is copied to a complementary RNA sequence
Translation: converts the RNA sequence to an amino acid sequence
DNA--> Transcription--> RNA --> Translation --> Polypeptide
Three types of RNA:
Messenger RNA (mRNA)-- a DNA sequence is copied to produce a complementary mRNA strand, which moves to a ribosome to be translated
The nucleotide sequence of the mRNA determines the sequence of amino acids in the polypeptide chain
Ribosomal RNA (rRNA), along with the proteins, makes up ribosomes; one catalyzes peptide bond formation
Transfer RNA (tRNA) binds to specific amino acids and to specific sequences on mRNA by base pairing
tRNA recognizes which amino acid should be added next in a growing polypeptide and carries it to the ribosome
Transcription requires several components
A DNA template for base pairings
The 4 nucleoside triphosphates (ATP, GTP, CTP, UTP)
An RNA polymerase enzyme--catalyzes synthesis of RNA from the DNA template.
RNA polymerases are processive-- a single enzyme--template binding results in polymerization of hundreds of RNA bases.
Unlike DNA polymerases, RNA polymerases do not need primers.
Transcription occurs in three steps:
Initiation requires a promoter--a control sequence of DNA that tells RNA polymerase where to start transcription and which strand to transcribe.
each promoter has a transcription initiation site -- where transcription begins
Other proteins (sigma factors and transcription factors) bind "upstream" from the initiation site and help RNA polymerase bind and determine which genes are expressed.
Elongation: RNA polymerase unwinds DNA and reads template in 3'-to-5' direction
RNA polymerase adds nucleotides to the 3' end of the new strand; the RNA transcript is anti-parallel to the DNA template strand
Uracil (not thymine) in the RNA molecule is paired with adenine in the DNA molecule
RNA polymerases moan proofread but allow o
Termination is specifically by a sequences of DNA sequence
Mechanism of termination are complex and vary among different genes and eukaryotes
In eukaryotes, multiple proteins are involved in recognizing the termination sequence and separation the newly formed RNA from the DNA template and RNA polymerase
Coding regions are sequences of DNA that are expressed as proteins
In prokaryotes, most of the DNA is made up of coding regions
In prokaryotes and viruses, several adjacent genes sometimes share one promoter
In eukaryotes each gene has its own promoter and most have noncoding sequences called introns
The transcribed regions are exons
Introns and exons both appear in the primary mRNA transcript (pre-mRNA); introns are removed from the final mRNA
Final RNA is the pre-mRNA without the introns
Nucleic Acid Hybridization
DNA slowly heated to denature, a probe with complementary base sequence is added to target DNA and form a double stranded hybrid molecule with each strand from a different source
Introns interrupt, but do not scramble, the DNA sequence of a gene
Most eukaryotic genes contain introns
The pre-mRNA is chemically modified:
RNA splicing removes introns and splices exons together
Consensus sequences are short sequences between exons and introns; they are first bound by snRNP's-- small nuclear ribonucleoprotein particles
The other proteins accumulate to form large RNA-protein complex called a spliceosome:
The complex cuts the pre-mRNA, releases the introns, and joins the exons together to produce mature mRNA
The pre-mRNA is also modified at both ends:
A 5' cap (G cap-- guanosine triphosphate) is added to the 5' end. It facilitates binding to a ribosome and prevents digestion by ribonuclease.
A poly A tail is added to the 3' end. It assists in export from the nucleus and contributes to the stability of the mRNA
Translation of the nucleotide sequence of an mRNA into an amino acid sequence occurs at ribosomes.
In prokaryotes, transcription and translation are coupled: ribosomes often bind to an mRNA as it is being transcribed.
In eukaryotes, the nuclear envelope separates mRNA production and translation.
*Key event--decoding mRNA nucleotide sequence into amino acid sequence
Codon: the genetic information is a series of sequential, non-overlapping, three-letter "words" (3 bases) called codons.
Each codon specifies an amino acid
*Genetic code broken in 1960's--20 amino acids encoded using the 4 nucleotides. Triplet codon could have 4 x 4 x 4= 64 variations
AUG- start codon- initiation signal for translation (methionine)
Stop Codon- UAA, UAG, UGA- termination for translation
Contains redundancies b/c more than one codon codes for AA
Genetic code is universal-used by all species on the planet
tRNAs link information in mRNA codons with specific amino acids.
Two events must occur:
tRNAs must read mRNA codons correctly
tRNAs must deliver the correct amino acids that correspond to each codon
For each amino acid, there is at least one specific type of tRNA.
The structure of tRNAs facilitates their three functions:
Bind to a specific amino acid and become “charged”
Bind to mRNA at the anticodon—a triplet that is complementary to the mRNA codon for the particular amino acid
Interact with the ribosomes non-covalently
Wobble: specificity for the base at the 3’ end of the codon is not always observed.
Example: Codons for alanine—GCA, GCC, and GCU—are recognized by the same tRNA
Wobble allows cells to produce fewer tRNA species but does not allow the genetic code to be ambiguous
tRNAs are charged by specific aminoacyl-tRNA synthetase
The large subunit has 3 tRNA binding sites:
A (amino acid) site binds with the anticodon of a charged tRNA
P (polypeptide) site is where tRNA adds its amino acid to the polypeptide
E (exit) site is where tRNA sits before being released from the ribosome
Ribosomes have a fidelity function: when proper binding occurs, hydrogen bonds form between the base pairs
Small subunit validates the match—if hydrogen bonds have not formed between all three base pairs, the tRNA is rejected
Translation also occurs in 3 steps:
Initiation complex consists of a charged tRNA and a ribosomal subunit both bound to the mRNA
Methionine-charged tRNA binds to AUG start codon
Large ribosomal subunit joins initiation complex with methionine-charged tRNA occupying the P site
Codon recognition: anticodon of incoming tRNA binds to codon at A site
Peptide bond formation: Proline is met by Methionine by peptidyl transferase activity of the large subunit
Elongation: free tRNA is moved to E site, releases, and codons shift over by one.
The release factor binds to the complex when a stop codon enters the A site
The release factor disconnects the polypeptide from the tRNA in the P site
The remaining components (mRNA and ribosomal subunits) separate
Several ribosomes can simultaneously translate a single mRNA molecule, producing multiple polypeptides at the same time.
A strand of mRNA with associated ribosomes is called a polyribosome, or polysome.
Proteins can undergo modifications both during and after translation
As a polypeptide emerges from the ribosome it may fold into its 3-D shape and perform its role in the cytosol
Or, polypeptides may contain a signal sequence that "targets" them to the nucleus, mitochondria, plastids, or peroxisomes
The signal sequence binds to a receptor protein on the organelle surface
A channel forms in the membrane and the protein moves into the organelle.
If a polypeptide has a signal directing it to the rough endoplasmic reticulum (RER), translation will pause, and the ribosome will bind to a receptor at the RER membrane
Then translation resumes, and as elongation continues, the polypeptide crosses into the RER lumen
It may be retained in the lumen, sent to other regions in the endomembrane system, or be secreted from the cellnucleotide sequence of an mRNA into an amino acid sequence occurs at ribosomes.
It may be ret