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Chapter 7 - From DNA to Protein
DNA to Protein
• DNA acts as a “manager” in the process of making proteins
• DNA is the template or starting sequence that is copied into RNA that is then used to make the protein
Central Dogma – Figure 7-1
• One gene – one protein
• This is the same for bacteria to humans
• DNA is the genetic instruction or gene
• DNA  RNA is called Transcription
– RNA chain is called a transcript
• RNA  Protein is called Translation
Expression of Genes – Figure 7-2
• Some genes are transcribed in large quantities because we need large amount of this protein
• Some genes are transcribed in small quantities because we need only a small amount of this protein
Transcription
• Copy the gene of interest into RNA which is made up of nucleotides linked by phosphodiester bonds – like
DNA
• RNA differs from DNA
– Ribose is the sugar rather than deoxyribose – ribonucleotides
– U instead of T; A, G and C the same
– Single stranded
• Can fold into a variety of shapes that allows RNA to have structural and catalytic functions
RNA Differences – Figures 7-3, 7-4 and 7-5
Transcription
• Similarities to DNA replication
– Open and unwind a portion of the DNA
– 1 strand of the DNA acts as a template
– Complementary base-pairing with DNA
• Differences
– RNA strand does not stay paired with DNA
• DNA re-coils and RNA is single stranded
– RNA is shorter than DNA
• RNA is several 1000 bp or shorter whereas DNA is 250 million bp long
Template to Transcripts – Figure 7-6
• The RNA transcript is identical to the NON-template strand with the exception of the T’s becoming U’s
RNA Polymerase – Figure 7-7
• Catalyzes the formation of the phosphodiester bonds between the nucleotides (sugar to phosphate)
• Uncoils the DNA, adds the nucleotide one at a time in the 5’ to 3’ fashion
• Uses the energy trapped in the nucleotides themselves to form the new bonds
RNA Elongation
• Reads template 3’ to 5’
• Adds nucleotides 5’ to 3’ (5’ phosphate to 3’ hydroxyl)
• Synthesis is the same as the leading strand of DNA
RNA Polymerase – Figure 7-8
• RNA is released so we can make many copies of the gene, usually before the first one is done
– Can have multiple RNA polymerase molecules on a gene at a time
Differences in DNA and RNA Polymerases
• RNA polymerase adds ribonucleotides not deoxynucleotides
• RNA polymerase does not have the ability to proofread what they transcribe
• RNA polymerase can work without a primer
• RNA will have an error 1 in every 10,000 nucleotides (DNA is 1 in 10,000,000 nucleotides)
Types of RNA (see Table 7-1)
• messenger RNA (mRNA) – codes for proteins
• ribosomal RNA (rRNA) – forms the core of the ribosomes, machinery for making proteins
• transfer RNA (tRNA) – carries the amino acid for the growing protein chain
DNA Transcription in Bacteria
• RNA polymerase must know where the start of a gene is in order to copy it
• RNA polymerase has weak interactions with the DNA unless it encounters a promoter
– A promoter is a specific sequence of nucleotides that indicate the start site for RNA synthesis
RNA Synthesis – Figure 7-9a
• RNA polymerase opens the DNA double helix and creates the template
• RNA polymerase moves nucleotide by nucleotide, unwinds the DNA as it goes
• Will stop when it encounters a STOP codon, RNA polymerase leaves, releasing the RNA strand
Sigma () Factor
• Part of the bacterial RNA polymerase that helps it recognize the promoter
• Released after about 10 nucleotides of RNA are linked together
• Rejoins with a released RNA polymerase to look for a new promoter
Start and Stop Sequences – Figure 7-9b
DNA Transcribed – Figure 7-10
• The strand of DNA transcribed is dependent on which strand the promoter is on
• Once RNA polymerase is bound to promoter, no option but to transcribe the appropriate DNA strand
• Genes may be adjacent to one another or on opposite strands
Eukaryotic Transcription – Figure 7-11
• Transcription occurs in the nucleus in eukaryotes, nucleoid region in bacteria
• Translation occurs on ribosomes in the cytoplasm
• mRNA is transported out of nucleus through the nuclear pores
RNA Processing – Figure 7-12
• Eukaryotic cells process the RNA in the nucleus before it is moved to the cytoplasm for protein synthesis
• The RNA that is the direct copy of the DNA is the primary transcript
• 2 methods used to process primary transcripts to increase the stability of mRNA being exported to the
cytoplasm
– RNA capping
– Polyadenylation
• RNA capping happens at the 5’ end of the RNA, usually adds a methylgaunosine shortly after RNA
polymerase makes the 5’ end of the primary transcript
• Polyadenylation modifies the 3’ end of the primary transcript by the addition of a string of A’s
Coding and Non-coding Sequences – Figure 7-13
• In bacteria, the RNA made is translated to a protein
• In eukaryotic cells, the primary transcript is made of coding sequences called exons and non-coding
sequences called introns
• It is the exons that make up the mRNA that gets translated to a protein
RNA Splicing – Figure 7-15
• Responsible for the removal of the introns to create the mRNA
• Introns contain sequences that act as cues for their removal
• Carried out by small nuclear riboprotein particles (snRNPs)
snRNPs – Figure 7-16
• snRNPs come together and cut out the intron and rejoin the ends of the RNA
• Intron is removed as a lariat – loop of RNA like a cowboy rope
Benefits of Splicing – Figure 7-18
• Allows for genetic recombination
– Link exons from different genes together to create a new mRNA
• Also allows for 1 primary transcript to encode for multiple proteins by rearrangement of the exons
Summary – Figure 7-19
RNA to Protein
• Translation is the process of turning mRNA into protein
• Translate from one “language” (mRNA nucleotides) to a second “language” (amino acids)
• Genetic code – nucleotide sequence that is translated to amino acids of the protein
Degenerate DNA Code – Figure 7-20
• Nucleotides read 3 at a time meaning that there are 64 combinations for a codon (set of 3 nucleotides)
• Only 20 amino acids
– More than 1 codon per AA – degenerate code with the exception of Met and Trp (least abundant AAs
in proteins)
Reading Frames – Figure 7-21
• Translation can occur in 1 of 3 possible reading frames, dependent on where decoding starts in the mRNA
Transfer RNA Molecules – Figure 7-22
• Translation requires an adaptor molecule that recognizes the codon on mRNA and at a distant site carries
the appropriate amino acid
• Intra-strand base pairing allows for this characteristic shape
• Anticodon is opposite from where the amino acid is attached
Wobble Base Pairing
• Due to degenerate code for amino acids some tRNA can recognize several codons because the 3 rd spot can
wobble or be mismatched
• Allows for there only being 31 tRNA for the 61 codons
Attachment of AA to tRNA
• Aminoacyl-tRNA synthase is the enzyme responsible for linking the amino acid to the tRNA
• A specific enzyme for each amino acid and not for the tRNA
2 ‘Adaptors’ Translate Genetic Code to Protein – Figures 7-23 and 7-26
Ribosomes – Figure 7-27
• Complex machinery that controls protein synthesis
• 2 subunits
– 1 large – catalyzes the peptide bond formation
– 1 small – binds mRNA and tRNA
• Contains protein and RNA
– rRNA central to the catalytic activity
• Folded structure is highly conserved
– Protein has less homology and may not be as important
• May be free in cytoplasm or attached to the ER
• Subunits made in the nucleus in the nucleolus and transported to the cytoplasm
Ribosomal Subunits – Figure 7-28
• 1 large subunit – catalyzes the formation of the peptide bond
• 1 small subunit – matches the tRNA to the mRNA
• Moves along the mRNA adding amino acids to growing protein chain
Ribosomal Movement – Figure 7-29
• 4 binding sites
– mRNA binding site
– Peptidyl-tRNA binding site (P-site)
• Holds tRNA attached to growing end of the peptide
– Aminoacyl-tRNA binding site (A-site)
• Holds the incoming AA
– Exit site (E-site)
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Step Elongation Phase – Figure 7-30
Elongation is a cycle of events
Step 1 – aminoacyl-tRNA comes into empty A-site next to the occupied P-site; pairs with the codon
Step 2 – C’ end of peptide chain uncouples from tRNA in P-site and links to AA in A-site
– Peptidyl transferase responsible for bond formation
– Each AA added carries the energy for the addition of the next AA
• Step 3 – peptidyl-tRNA moves to the P-site; requires hydrolysis of GTP
– tRNA released back to the cytoplasmic pool
Initiation Process – Figure 7-32
• Determines whether mRNA is synthesized and sets the reading frame that is used to make the protein
• Initiation process brings the ribosomal subunits together at the site where the peptide should begin
• Initiator tRNA brings in Met
– Initiator tRNA is different than the tRNA that adds other Met
Ribosomal Assembly Initiation Phase
• Initiation factors (IFs) catalyze the steps – not well defined
• Step 1 – small ribosomal subunit with the IF finds the start codon –AUG
– Moves 5’ to 3’ on mRNA
– Initiator tRNA brings in the 1st AA which is always Met and then can bind the mRNA
• Step 2 – IF leaves and then large subunit can bind – protein synthesis continues
• Met is at the start of every protein until post-translational modification takes place
Eukaryotic vs Procaryotic – Figure 7-12
• Procaryotic
– No CAP; have specific ribosome binding site upstream of AUG
– Polycistronic – multiple proteins from same mRNA
• Eucaryotic
– Monocistronic – one polypeptide per mRNA
Protein Release – Figure 7-34
• Protein released when a STOP codon is encountered
– UAG, UAA, UGA (must know these sequences!)
• Cytoplasmic release factors bind to the stop codon that gets to the A-site; alters the peptidyl
transferase and adds H2O instead of an AA
• Protein released and the ribosome breaks into the 2 subunits to move on to another mRNA
Polyribosomes – Figure 7-35
• As the ribosome moves down the mRNA, it allows for the addition of another ribosome and the start of
another protein
• Each mRNA has multiple ribosomes attached, polyribosome or polysome
Regulation of Protein Synthesis
• Lifespan of proteins vary, need method to remove old or damaged proteins
• Enzymes that degrade proteins are called proteases – process is called proteolysis
• In the cytosol there are large complexes of proteolytic enzymes that remove damaged proteins
• Ubiquitin, small protein, is added as a tag for disposal of protein
Protein Synthesis
• Protein synthesis takes the most energy input of all the biosynthetic pathways
• 4 high-energy bonds required for each AA addition
– 2 in charging the tRNA (adding AA)
– 2 in ribosomal activities (step 1 and step 3 of elongation phase)
Summary – Figure 7-37
Ribozyme – Figure 7-40; Table 7-3
• A RNA molecule can fold due to its single stranded nature and in folding can cause the cleavage of other
RNA molecules
• A RNA molecule that functions like an enzyme hence ribozyme name