Chapter 17 - from gene to protein

CHAPTER 17 FROM GENE TO PROTEIN The The information information content content of of genes genes is is in in the the form form of of specific specific sequences sequences of of nucleotides nucleotides along along the the DNA DNA strands. strands. The The DNA DNA of of an an organism organism leads leads to to specific specific traits traits by by dictating dictating the the synthesis synthesis of of proteins proteins and and of of RNA RNA molecules molecules involved involved in in protein protein synthesis synthesis (gene (gene expression.) expression.) 1 ARCHIBALD GARROD The The study study of of metabolic metabolic defects defects provided provided

evidence evidence that that genes genes specify specify proteins. proteins. Garrod Garrod discovered discovered that that proteins proteins (enzymes) (enzymes) are are the the link link between between genotype genotype and and phenotype. phenotype. He He figured figured out out that that some some diseases diseases are are the the inability inability He He noticed noticed that that the the diaper diaper of of a a baby baby was was very very brown. brown. He He determined determined that that the the baby baby had had alkaptonuria, alkaptonuria, which which is is a a recessively recessively inherited inherited disorder disorder where where the the urine urine is is a

a brown brown color. color. This This is is due due to to homogentisic homogentisic acid acid which which cannot cannot be be broken broken down down in in the the body, body, so so it it is is excreted excreted in in the the urine. urine. The The reason reason it it cannot cannot be be broken broken down down is is because because there is an absence of the enzyme needed inherited inherited to to make make enzymes enzymes 2 Beadle and Tatum showed the relationship between genes and enzymes. They used the bread mold Neurospora and exposed it to X-rays to get mutants. They found 3 different classes of mutants. Each mutant was defective in a different gene. They exposed these mutants to different environments to see which ones allowed arginine to grow. They deduced that each mutant was unable to carry out one step in the arginine pathway probably because it lacked the necessary enzyme BEADLE AND TATUM 3

ONE GENE ONE ENZYME HYPOTHESISAND THE EVOLUTION OF THAT HYPOTHESIS From From Beadle Beadle and and Tatums Tatums experiments, experiments, they they came came up up with with the the one one gene, gene, one one enzyme enzyme hypothesis. hypothesis. However, not all proteins are enzymes, so it became the one genegene- one one protein protein hypothesis. hypothesis. BUTsome BUTsome genes genes have have more more than than one one polypeptide polypeptide (THINK: (THINK: quaternary quaternary structure structure of of proteins), proteins), so so it it led led to to the the one one genegene- one one polypeptide polypeptide hypothesis. hypothesis. The The newest newest discoveries discoveries have have been been taken taken into into consideration consideration and and the the scientific scientific community community have have updated updated the

the definition definition of of a a gene gene as: as: A A gene gene is is a a region region of of DNA DNA that that can can be be expressed expressed to to produce produce a a final final functional functional product product that that is is either either a a polypeptide or an RNA molecule. 4 OVERVIEW: TRANSCRIPTION TRANSLATION DNA DNA translation translation RNA processing Primary Primary transcript transcript (pre-mRNA) (pre-mRNA) RNA processing mRNA mRNA protein protein Genes Genes provide provide the the instructions instructions for for making making specific specific proteins proteins and and getting getting from from gene

gene to to protein protein needs needs two two stages: stages: transcription transcription Transcription Transcription = = DNA DNA RNA RNA Translation Translation = = RNA RNA Protein Protein 5 TRANSCRIPTION AND TRANSLATION IN PROKARYOTES VS. EUKARYOTES The The basic basic mechanics mechanics of of transcription transcription and and translation translation are are similar similar in in eukaryotes eukaryotes and and bacteria. bacteria. Bacteria Bacteria lack lack nuclei, nuclei, and and their their DNA DNA is is not not separated separated from

from ribosomes ribosomes and and other other protein-synthesizing protein-synthesizing equipment. equipment. This allows the coupling This allows the coupling of of transcription transcription and and translation. translation. In In a a eukaryotic eukaryotic cell, cell, transcription transcription occurs occurs in in the the nucleus, nucleus, and and translation translation occurs occurs at at ribosomes ribosomes in in the the cytoplasm. cytoplasm. The The molecular molecular chain chain of of command command in in a a cell cell has has a a directional directional flow flow of of genetic genetic information: information: DNA DNA RNA RNA protein protein Francis Francis Crick

Crick dubbed dubbed this this concept concept the the central central dogma dogma in in 1956. 1956. 6 CODING FOR AMINO ACIDS The The message message is is carried carried in in RNA RNA in in the the form form of of codons codons (3 (3 bases). bases). It It is is read read in in the the 5 5 3 3 direction. direction. 7 With With a a triplet triplet code, code, three three consecutive consecutive bases bases specify

specify an an amino amino acid, acid, creating creating 4 433 (64) (64) possible possible code code words. words. During During transcription, transcription, one one DNA DNA strand, strand, the the template template strand, strand, provides provides a a template template for for ordering ordering the the sequence sequence of of nucleotide nucleotide bases bases in in an an mRNA mRNA transcript. transcript. The The mRNA mRNA base base triplets triplets are are called called codons. codons. Each Each codon codon specifies specifies which which one one of of the the 20 20 amino amino acids acids will will be be incorporated

incorporated at at the the corresponding corresponding position position along along a a polypeptide polypeptide chain. chain. The The starting starting point point establishes establishes the the reading reading frame; frame; subsequent subsequent codons codons are are read read in in groups groups of of three three nucleotides. nucleotides. THE TRIPLETS (CODONS) CODE FOR THE SPECIFIC AMINO ACIDS 8 The The genetic genetic code code must must have have evolved evolved very very early early in in the the history history of of life life It It is is nearly nearly universal, universal, shared shared by

by organisms organisms from from the the simplest simplest bacteria bacteria to to the the most most complex complex plants plants and and animals. animals. Nirenberg Nirenberg determined determined the the first first match: match: UUU UUU codes codes for for the the amino amino acid acid phenylalanine. phenylalanine. Sixty-one Sixty-one of of 64 64 triplets triplets code code for for amino amino acids. acids. Marshall Nirenberg deciphered the code for the amino acids in 1961. The The codon codon AUG AUG not not only only codes codes for for the the amino amino acid acid methionine methionine but but also also indicates

indicates the the start start or or initiation initiation of of translation. translation. Three Three codons codons do do not not indicate indicate amino amino acids acids but but are are stop stop signals signals 9 TRANSCRIPTIO N DNA DNA RNA RNA Promoter Promoter DNA DNA sequence sequence where where RNA RNA attaches attaches and and initiates initiates transcription transcription Terminator Terminator sequence sequence that that signals signals the the end end of of transcription transcription Transcription Transcription Unit Unit sequence sequence of of DNA DNA that that is is transcribed transcribed into

into RNA RNA 3 3 Steps Steps of of Transcription: Transcription: 1. 1. Initiation Initiation 2. 2. Elongation Elongation 3. 3. Termination Termination 10 TRANSCRIPTION - INITIATION The promoter determines which strand is the template and then transcription factors help RNA polymerase bind. The TATA box is an important part of the promoter that helps initiate transcription. The transcription complex consists of the promoter, transcription factors, and RNA polymerase. RNA RNA polymerase polymerase separates separates the the DNA DNA strands strands at at the the appropriate appropriate point point and and joins joins RNA RNA nucleotides nucleotides complementary complementary to to the the DNA DNA template template strand. strand. Like Like DNA DNA polymerases, polymerases, RNA RNA polymerases polymerases can

can assemble assemble a a polynucleotide polynucleotide only only in in its its 53 53 direction direction (therefore (therefore the the 11 template template strand strand is is 3 3 5.) 5.) TRANSCRIPTIO NELONGATION The The RNA RNA polymerase polymerase adds adds RNA RNA nucleotides nucleotides about about 101020 20 at at a a time time to to the the growing growing 3 3 end. end. Several Several mRNA mRNA strands strands can can be be made made at at the the same same time.several time.several different different RNA RNA polymerases polymerases can can all all be be on

on the the same same DNA DNA molecule molecule and and can can all all create create mRNA. mRNA. This This helps helps the the cell cell make make the the encoded encoded protein protein in in large large 12 amounts. amounts. TRANSCRIPTION TERMINATION In In prokaryotes, prokaryotes, termination termination stops stops at at the the termination termination signal signal (end (end of of the the gene) gene) In In eukaryotes, eukaryotes, transcription transcription continues continues for for 10-35 10-35 nucleotides nucleotides past past the the stop stop signal. signal. Later Later in in the the process, process, it it gets

gets cut cut down. down. At At this this point, point, transcription transcription has has given given us us the the primary primary transcripts transcripts or or prepremRNA mRNA 13 RNA PROCESSING: MODIFYING THE PRE-MRNA --- At At the the 5 5 end, end, a a 5 5 cap cap is is added added (which (which is is a a modified modified guanine guanine molecule) molecule) At At the the 3 3 end, end, there there is is the the poly-A poly-A tail tail (50-250 (50-250 adenine adenine nucleotides) nucleotides) functions functions in in helping helping to to inhibit inhibit degradation degradation and and helps helps exportation exportation from

from nucleus) nucleus) ---- Both Both of of these these modifications modifications have have several several important important functions: functions: -- Exporting Exporting mRNA mRNA from from the the nucleus nucleus -- Protecting Protecting mRNA mRNA from from hydrolytic hydrolytic enzymes enzymes -- Helping Helping the the ribosome ribosome attach attach to to the the 5 5 end end of of the the mRNA mRNA After After both both ends ends are are modified, modified, the the introns introns (non(noncoding coding portions) portions) are are spliced spliced out. out. 14 Introns Introns = = non-coding non-coding segments segments Exons

Exons = = coding coding segments segments RNA SPLICING The The introns introns are are cut cut out out using using splicesomes. splicesomes. Therefore, Therefore, the the mRNA mRNA that that leaves leaves the the nucleus nucleus (exons (exons only) only) is is the the abridged abridged version version that that only only carries carries genes genes not not filler filler DNA. DNA. 15 RNA SPLICING TECHNIQUE SPLICESOME There There are are short short sequences sequences S at at the the end end of of introns introns that that signal

signal to to the the snRNPs snRNPs (small (small nuclear nuclear ribonucleoproteins). ribonucleoproteins). The The snRNPs snRNPs recognize recognize these these sites sites and and the the splicesomes splicesomes then then cut cut out out the the introns introns and and reattach reattach the the exons. exons. Ribozyme Ribozyme RNA RNA molecules molecules that that act act like like enzymes; enzymes; in in some some organsisms organsisms RNA RNA splicing splicing can can occur occur without without additional additional proteins proteins because because the the introns introns can can catalyze catalyze their their own own excision excision 16

ALTERNATIVE RNA SPLICING Humans Humans can can get get along along with with a a small small number number of of genes genes because because we we can can shuffle shuffle our our DNA; DNA; different different polypeptides polypeptides can can be be made made depending depending on on which which segments segments we we consider consider introns introns and and which which are are considered considered exons. exons. 17 TRANSLATION FROM RNA TO PROTEIN BUILDING A POLYPEPTIDE! 18 tRNA - TRANSLATOR -The cell is always stocked with all 20 AAs (from diet) -The tRNA is folded like a cloverleaf; on one side it has an anticodon that matches up with the codon from the mRNA; on the other side it carries a

specific AA -The tRNAs are used over and over; they rd rd Wobble base pairing; sometimes their AAs Wobble relaxation relaxation of of 3 3rddrop baseoff pairing; sometimes the the 3 3rd base base of of the the ANTICODON which is and then go get ANTICODON has has an an I I (inosine), (inosine), which is an an altered altered adenine; adenine; this this can can match each had another to be used match up up with with U, U, C, C, or or A; A; If If each anticodon anticodon had to to be be a a perfect perfect match match to to each to

again each codon, codon, we we would would expect expect to find find 61 61 types types of of tRNA, tRNA, but but the the actual actual number of tRNAs more number is is about about 45, 45, because because the the anticodons anticodons of some some tRNAs recognize recognize more CCI anticodon can match up with CCI anticodon can match up with than than one one codon codon (the (the wobble!!) wobble!!) 19 GGU, GGU, GGC GGC and and GGA GGA (codons) (codons) AMINOACYLtRNA SYNTHETASE This This enzyme enzyme attaches attaches each each AA

AA to to its its appropriate appropriate tRNA. tRNA. This This process process uses uses 1 1 ATP ATP There There are are 20 20 different different aminoacyl-tRNA aminoacyl-tRNA synthetases synthetases (one (one for for each each AA) AA) Process: Process: 1. 1. The The active active site site of of the the aminoacyl aminoacyl tRNA tRNA synthetase synthetase binds binds to to the the AA AA and and ATP ATP 2. 2. The The ATP ATP loses loses 2 2P P groups groups to to become become AMP AMP and and binds binds with with the the AA AA 3. 3. Then

Then the the right right tRNA tRNA binds binds to to the the AA AA and and displaces displaces the the AMP AMP 4. 4. The The enzyme enzyme then then releases releases the the 20 RIBOSOMES: SITES OF TRANSLATION Ribosomes Ribosomes consist consist of of 2 2 subunits, subunits, large large and and small; small; they they are are composed composed of of rRNA rRNA and and proteins proteins They They have have 3 3 binding binding sites sites for for tRNA: tRNA: E E= = about about to to exit exit P P= = holds holds the the AA AA chain chain

A A= = on-deck on-deck AA AA The The ribosomes ribosomes itself itself catalyzes catalyzes the the peptide peptide bond bond between between amino amino acids. acids. Like Like transcription, transcription, translation translation can can be be divided divided into into 3 3 stages: stages: -- initiation initiation -- elongation elongation -- termination termination 21 ENERGY SOURCE FOR TRANSLATION GTP GTP (guanosine (guanosine triphosphate) triphosphate) energy energy source source for for translation; translation; this this is is very very similar similar to to ATP ATP and and releases releases energy energy by by breaking breaking off

off phosphates phosphates 22 TRANSLATION INITIATION Steps: Steps: 1. 1. Small Small ribosomal ribosomal subunit subunit binds binds to to mRNA mRNA leader leader (5 (5 end) end) 2. 2. Initiator Initiator tRNA tRNA (methionine) (methionine) binds binds to to start start codon AUG codon AUG 3. 3. Next Next the the large large ribosomal ribosomal subunit subunit binds binds 4. 4. All All of of these these components components (small (small unit, unit, mRNA, mRNA, tRNA, tRNA, large large subunit) subunit) are are brought brought together together by by initiation initiation factors factors and and form form the the translation

translation initiation initiation complex complex 23 TRANSLATION ELONGATION 3 3 step step process process for for each each AA: AA: 1. 1. Codon Codon recognition recognition 2. 2. Peptide Peptide bond bond formation formation 3. 3. Translocation Translocation This This process process uses uses elongation elongation factors factors (proteins) (proteins) 24 TRANSLATION - TERMINATION When When a a stop stop codon codon (mRNA) (mRNA) gets gets to to get get the the A-site A-site and and instead instead of of a a tRNA tRNA binding, binding, a a release release factor

factor binds. binds. This This adds adds a a water water molecule molecule to to the the AA AA chain, chain, and and then then releases releases the the chain chain from from the the ribosome. ribosome. After After the the chain chain is is released, released, all all the the factors factors dissociate dissociate from from one one another. another. 25 OVERVIEW PROTEIN SYNTHESIS 26 POLYRIBOSOMES OR POLYSOMES This This is is when when many many ribosomes ribosomes trail trail along along the the same same mRNA mRNA molecule. molecule. They They can can translate translate many

many proteins proteins simultaneously simultaneously and and therefore therefore are are much much more more efficient. efficient. 27 POST-TRANSLATIONAL MODIFICATIONS During and after synthesis, a polypeptide spontaneously coils and folds to its three-dimensional shape. In addition, proteins may require post-translational modifications before doing their particular job. These modifications may require additions such as sugars, lipids, or phosphate groups to amino acids. In other cases, a polypeptide may be cleaved in two or more pieces OR two or more polypeptides may join to form a protein with quaternary structure. 28 SIGNAL PEPTIDES DETERMINE WHETHER RIBOSOME WILL BE ATTACHED OR FREE All All ribosomes ribosomes start start as as free free (in (in the the cytosol); cytosol); however, however, the the polypeptide polypeptide can can cue cue the the ribosome ribosome to to go go attach attach to to the the ER ER and and become become bound. bound. The The signal signal peptide peptide is

is a a sequence sequence of of about about 20 20 AAs AAs near near the the front front of of the the strand strand that that tells tells the the ribosome ribosome to to go go attach. attach. This This is is the the case case for for proteins/ proteins/ enzymes enzymes that that are are going going to to be be secreted secreted from from the the cell. cell. The The signal signal recognition recognition particle particle (SRP) (SRP) sees sees this this signal signal peptide peptide and and brings brings the the ribosome ribosome to to the the ER ER to to attach. attach. 29 MUTATIONS Mutations Mutations are are changes

changes in in the the genetic genetic material material of of a a cell cell (or (or virus). virus). They They are are the the ultimate ultimate source source of of new new genes genes (and (and genetic genetic diversity!) diversity!) A A point point mutation mutation (also (also called called a a substitution) substitution) is is a a change change in in one one base base pair. pair. It It can can have have huge huge effects effects (sickle (sickle cell) cell) or or no no affect affect at at all all (silent (silent mutation), mutation), depending depending on on which which base base is is affected affected and and where where the

the AA AA is is located located in in the the protein. protein. 30 Missense Missense = = codes codes for for a a different different AA AA Nonsense Nonsense = = changes changes into into a a stop stop codon, codon, so so it it leads leads to to a a nonfunctional nonfunctional protein protein Silent Silent = = changes changes the the nucleotide nucleotide but but it it codes codes for for the the same same AA AA MISSENSE AND NONSENSE MUTATIONS 31 FRAMESHIFT MUTATIONS A frameshift mutation A frameshift mutation is is when when there

there is is an an insertion insertion or or deletion deletion that that causes causes the the reading reading frame frame to to change. change. This This means means that that all all of of the the AAs AAs after after the the mutation mutation will will be be wrong. wrong. It It has has disastrous disastrous effects. effects. 32 MUTATIONS CAN OCCUR DURING DNA REPLICATION, DNA REPAIR, OR DNA RECOMBINATION Errors during DNA replication or recombination can lead to nucleotide-pair substitutions, insertions, or deletions. Mutagens are chemical or physical agents that interact with DNA to cause mutations. Physical agents include high-energy radiation like X-rays and ultraviolet light. Chemical mutagens cause mutations in different ways. 33

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