Instructions in DNA direct synthesis of proteins.

Instructions in DNA direct synthesis of proteins. There are many genes on each chromosome, and each gene codes for one polypeptide.

Neurospora is a bread mold that can survive on minimal media (salts, sucrose and biotin). It uses a multistep pathway to make the amino acid, arginine. After exposing Neurospora to X-rays, mutants that could not make arginine were isolated.

Each mutant was defective at a different step of the pathway. This was the first evidence of the one gene-one enzyme hypothesis.

The Central Dogma: DNA mRNA Protein Transcription—DNA mRNA Translation—mRNA Protein

There are 3 kinds of RNA involved in translation: • mRNA—”messenger”—made from a DNA template and codes for polypeptides • 2. tRNA—”transfer”—used in translation to read the mRNA code and add amino acids to the polypeptide • 3. rRNA—”ribosomal”—component of ribosomes

There are 4 different nucleotides but 20 different amino acids. Each 3-nucleotide sequence in mRNA is called a CODON and specifies one amino acid. There are 43 combinations of nucleotides, so that means that there are 64 codons for 20 amino acids. The genetic code is redundant – more than 1 codon can code for the same amino acid But it is NOT ambiguous! Each codon codes for only 1 amino acid.

TRANSCRIPTION The 3’  5’ strand of DNA is called the template. mRNA is made in the 5’  3’ direction. TRANSLATION Codons are read in the 5’  3’ direction to determine amino acid sequence.

There is one “start” codon and 3 “stop” codons. “Start” also codes for methionine. Codons are the sequences in mRNA, NOT DNA!

Abbreviations for the 20 amino acids Ala = alanine (A) Arg = arginine (R) Asn = asparagine (N) Asp = aspartic acid (D) Cys = cysteine (C) Gln = glutamine (Q) Glu = glutamic acid (E) Gly = glycine (G) His = histadine (H) Ile = isoleucine (I) Leu = leucine (L) Lys = lysine (K) Met = methionine (M) Phe = phenylalanine (F) Pro = proline (P) Ser = serine (S) Thr = threonine (T) Trp = tryptophan (W) Tyr = tyrosine (Y) Val = valine (V)

The reading frame is non-overlapping mRNA: UGGUUUGGCCGUUUU polypeptide:Trp—Phe—Gly—Arg—Phe The genetic code is shared nearly universally among living organisms!

Transcription: making mRNA from a DNA template • Initiation—Specific DNA sequences called promoters mark where initiation begins • Elongation—RNA polymerase unwinds DNA strands and adds RNA nucleotides in the 5’3’ direction • Termination—RNA polymerase falls off at the termination sequence

Prokaryotes have one RNA polymerase. It reads the template strand of DNA and then the mRNA peels away from the template as it’s made.

Eukaryotic transcription is more complicated. There are 3 types of RNA polymerases involved. RNA polymerase II is used to make mRNA. Transcription factors help RNA pol II recognize the promoter by binding to the TATA box.

Prokaryotic mRNA is ready for translation as soon as it leaves the DNA template, but eukaryotic mRNA needs to be processed before it leaves the nucleus to be translated. The 5’ cap and 3’ poly-A tail help export the mRNA out of the nucleus, protect the mRNA from degradation, and help the ribosome recognize where to bind. UTR—untranslated region—doesn’t code for anything

Exons are coding sequences in mRNA, and introns are non-coding sequences. The introns must be spliced out by spliceosomes that are made up of snRNPs (small nuclear ribonucleoproteins) and proteins.

RNA splicing removes intronsand joins exons together to make mRNA that’s ready to be translated.

Each exon corresponds to one polypeptide domain

Translation: making protein from an mRNA template Protein synthesis occurs as the mRNA slides through the ribosome. tRNAs add the correct amino acids to the growing polypeptide chain.

tRNAs are transcribed from DNA and have a looped, “t” shaped structure.

The cytoplasm contains tRNAs for all 20 amino acids. The anticodon is complementary to the mRNA codon. GAU anticodon tRNA: mRNA: CUA codon

There should be 61 different tRNAs. However, there are only about 45. Base pairing rules at third base are not as strict as at the other bases. At the third base, U can pair with A or G! So ACA and ACG both code for Thr. One tRNAcan be used for both codons! (Anticodon = UGU)

The relaxation of base-pairing rules is called WOBBLE. Inosine (I) is a modified base that pairs with U, C, or A. GGU, GGC, and GGA all code for glycine, so there only needs to be one tRNA with the anticodonCCI to match any of the three codons. U U A G G

Aminoacyl-tRNAsynthetases addthe correct amino acids to the appropriate tRNAs. There are 20 aminoacyl-tRNAsynthetases—one for each amino acid! Attaching the amino acid requires ATP.

Ribosomeshelp tRNAanticodonsrecognize their complementary codonson mRNA. Each ribosomal subunit is made up of proteins and rRNA. Ribosomesare made in the nucleolus and then are exported to the cytosol.

Translation Initiation • Small ribosomal subunit binds to mRNA • 2. tRNA with UAC anticodonbinds to AUG start codon • 3. Large ribosomal subunit binds to mRNA so that • the initiator tRNA is in the P site • 4. One GTP is used!

Elongation

Termination There are no tRNAs that match stop codons! A release factor binds to the stop codon. Water is added to break the bond between the last amino acid and its tRNA.

Polyribosomes= multiple ribosomes work to translate the same mRNA simultaneously

Proteins may need post-translational modifications before they can function correctly. • attachment of sugars, lipids, or phosphates • removal of amino acids from leading end of polypeptide • activation by cleavage into 2 or more pieces • joining together of 2 or more subunits

How proteins get secreted • start on free ribosomes • SRP binds to signal peptide • ribosome goes to ER 4. SRP is released 5. signal peptide is removed 6. protein is released INSIDE ER membrane.

In prokaryotes, transcription and translation are coupled.

Mutations in DNA affect proteins Point mutations involve a change in one base pair. A single substitution causes sickle cell anemia.

Instructions in DNA direct synthesis of proteins.