Chapter 6 How Cells Read the Genome: From DNA to Protein RNA Transcription RNA processing Translation

RNA contains Ribose and Uracil DNA contains Deoxyribose and Thymine Phosphodiester bonds between RNA and DNA are the same

RNA is single stranded, thus it can fold into specific structures by base-pairing with complementary sequences

TRANSCRIPTION: Promoter RNA Polymerase Recognize initiation site Separation of DNA Base pairing Energy requiring process No primer required Process begins at specific site within the promoter

RNA polymerase: DNA is transcribed by RNA polymerase No primer required Small window is made in DNA approximately 9 bases in length

MOVIE

RNA polymerase can move in either direction

RNA polymerase uses the antisense strand as a template to produce primary RNA strand

Genes can be expressed with different efficiencies

RNA polymerase requires a protein complex at a promoter to initiate RNA synthesis

Enhancer binding proteins can act from a distance to enhance initiation of transcription.

RNA Processing: Prokaryotes: transcription and translation can be concurrent. Eukaryotes: Nucleus (RNA synth) and cytoplasm (Prot synth) are separated. Primary transcript undergoes several modifications. 5’ cap is added to 5’ nucleotide; m7Gppp (Stability) String of adenylic acids are added to the 3’ end (Poly A tail) Splicing: internal cleavage to excise introns followed by ligation of coding exons

5’-methyl cap of mRNAs Once 25 nucleotides are synthesized the 5’ end of the primary transcript is modified. Used to ID mRNA and protect from degradation.

Polycistronic Monocistronic Organization of Genes differ in prokaryotes and eukaryotes: Gene: Unit of DNA that contains the info to specificy synthesis of a single polypeptide Compact, colinear mRNA Diff chromosomes; one mRNA Prokaryotes (operon) Eukaryotes Trp mRNA 5’ 3’ Proteins Polycistronic Monocistronic

Eukaryotes: Organization of DNA within a single gene; Exons and introns Primary transcript Exon Intron Exon Intron Primary transcript Exon Intron Exon Intron modification Exon Exon Splicing Cap poly A tail RNA PROCESSING

The structure of two human genes

RNA splicing A specific A attacks the 5’ splice site Upon cleavage the free OH group reacts with the next exon sequence releasing the intron

The primary transcript can be spliced into several distinct mRNAs Alternative splicing The primary transcript can be spliced into several distinct mRNAs

Spliced and polyA mRNA is exported through the nuclear pore

TRANSLATION The three roles of RNA in protein synthesis messenger RNA (mRNA). This will later be translated into a polypeptide. (Carries information in the form of a three-base code) transfer RNA (tRNA). RNA molecules that carry amino acids to the growing polypeptide. (Is key to deciphering the code) ribosomal RNA (rRNA). This will be used in the building of ribosomes: machinery for synthesizing proteins by translating mRNA. (physically move along the mRNA molecule, catalyze assembly of a.a. into prot.

Genetic information is carried as three base genetic code Four bases (A G C T/U) must encode for 20 a.a. Therefore a combination is required: 43 = 64 Triplet code is called a CODON that must begin at a precise site Of 64, 61 specify individual a.a.; and three are STOP codons starting codon is AUG (methionine) Code is universal, synonymous, degenerate Reading frame 3rd base in codon “wobble” frameshifts/deletions/insertions (MUTATIONS)

The genetic code

Three different reading frames

The decoding function and process: (tRNA and aminoacyl tRNA synthases) 3D structure important for function The acceptor stem includes the 5' and 3' ends of the tRNA. The 5' end is generated by RNase P. The 3' end is the site which is charged with amino acids for translation. Some aminoacyl tRNA synthetases interact with both the acceptor 3' end and the anticodon when charging tRNAs. Each tRNA is recognized by only one synthase; tRNA > a.a. ; tRNA can attach to more than one codon (basis for wobble in genetic code)

How do you charge a tRNA with an aa

Several different views of a tRNA

The wobble position is found at the third base of the codon

Polypeptide synthesis is a processive reaction

Ribosomes are the protein synthesizing machinery: Composed of rRNA and more than 50 proteins Small and large subunit; present in cytosol Similar 3D structure across species

The ribosome is a ribonucleoprotein

mRNA Translation Step: MOVIES Basepair to codon, A site A new peptide is formed Ejecting spent tRNA and resetting for next round MOVIES

The Steps of Translation: 1. Initiation: The small subunit of the ribosome binds to a site "upstream“ (on the 5' side) of the start of the message. It proceeds downstream (5' -> 3') until it encounters the start codon AUG. Here it is joined by the large subunit and a special initiator tRNA. The initiator tRNA binds to the P site (shown in pink) on the ribosome. In eukaryotes, initiator tRNA carries methionine (Met). (Bacteria use a modified methionine designated fMet.) 2. Elongation: An aminoacyl-tRNA (a tRNA covalently bound to its amino acid) able to base pair with the next codon on the mRNA arrives at the A site (green) associated with: an elongation factor (called EF-Tu in bacteria) GTP (the source of the needed energy). The preceding amino acid (Met at the start of translation) is covalently linked to the incoming amino acid with a peptide bond (shown in red). The initiator tRNA is released from the P site. The ribosome moves one codon downstream. This shifts the more recently-arrived tRNA, with its attached peptide, to the P site and opens the A site for the arrival of a new aminoacyl-tRNA. This last step is promoted by another protein elongation factor (named EF-G) and the energy of another molecule of GTP.

Note: the initiator tRNA is the only member of the tRNA family that can bind directly to the P site. The P site is so-named because, with the exception of initiator tRNA, it binds only to a peptidyl-tRNA molecule; that is, a tRNA with the growing peptide attached. The A site is so-named because it binds only to the incoming aminoacyl-tRNA; that is the tRNA bringing the next amino acid. So, for example, the tRNA that brings Met into the interior of the polypeptide can bind only to the A site. 3. Termination: The end of the message is marked by one or more STOP codons (UAA, UAG, UGG). No tRNA molecules have anticodons for STOP codons. However, a protein release factor recognizes these codons when they arrive at the A site. Binding of this protein releases the polypeptide from the ribosome. The ribosome splits into its subunits, which can later be reassembled for another round of protein synthesis. Polysomes A single mRNA molecule usually has many ribosomes traveling along it, in various stages of synthesizing the polypeptide. This complex is called a polysome.

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DNA to Protein

Each step in the synthesis of a protein can ultimately affect the amount of protein found in a cell