With the complete nucleotide sequence of the genome in a variety of organisms, geneticists can consult the genetic code (define) to decide what parts of a genome are likely to be _______. As a result, modern geneticists can discover the number and amino acid sequences of all the polypeptides that determine _______
- genetic code: the cipher equating nucleotide sequence with amino acid sequence
The cellular mechanisms that carry out gene expression (define) can be interpreted as phenotype. As intricate as some of the details may appear, the general scheme of gene expression is elegant and straightforward (explain)
- gene expression: the means by which genetic information is converted into RNA and then possibly into a polypeptide
- explanation: within each cell, genetic information flows from DNA to RNA to protein
The genetic flow from DNA to RNA to protein was set forward as the _______ ________ of molecular biology by Francis Crick in 1957. As Crick explained, " Once information has passed in to _______, it cannot get out again."
The Central Dogma maintains that genetic information flows in two distinct stages (2-story)
- The conversion of the information in DNA to its equivalent in RNA is known as transcription In the second stage of gene expression, the cellular machinery decodes the sequence of nucleotides in mRNA into a sequence of amino acids (a polypeptide) by the process known as translation
Transcript: The product of transcription, it is a molecule of messenger RNA (mRNA)
Translation takes place on molecular workbenches called _______ which are composed of _______ and _______ ________
- proteins and ribosomal RNAs (rRNAs)
Translation depends on the dictionary known as the ______ _____, which defines each amino acid in terms of specific sequences of ______ nucleotides
Translation also requires transfer RNAs (tRNAs). What are they?
tRNAs: small RNA adapter molecules that place specific amino acids at the correct position in a growing polypeptide chain
The Central Dogma does not explain the behavior of all genes. As Crick realized, many genes are _______ into RNAs that are never _________ into protein.
What about nucleotide bases is the key to the transfer of information from DNA to RNA, and from RNA to protein?
The ________ (directionally) of DNA, RNA, and polypeptides help guide the mechanisms of gene expression
- The pairing of complementary bases
Like DNA replication and recombination, gene expression requires an input of _______ and the participation of specific _______, ______, and macromolecular assemblies, such as _______.
________ that change genetic information or obstruct the flow of its expression can have dramatic effects on phenotype
- proteins, RNAs...
The language of nucleic acids is written in four nucleotides in the DNA dialect (name them); in the RNA dialect (name them); while the language of proteins is written in _____ _____
- DNA: A G C T
- RNA: A G C U
- amino acids
What is needed to get to each step 2-story
- RNA polymerase copies from one strand of the DNA double helix.
- Ribosomes translate the DNA code to the appropriate amino acid sequence for the polypeptide.
- *RNA may be processed before translation.
Researchers reasoned that if only one nucleotide represented an amino acid, there would be information for only four amino acids: A would encode _____ amino acid; G the same and so on. If two nucleotides represented each amino acid, there would be ________ possible combinations of couplets.
Of course, if the code consisted of groups containing one or two nucleotides, it would have _____ groups and could account for all the amino acids, but there would be nothing left over to signify the ______ required to denote where one group ends and the next begins
Groups of three nucleotides in a row would provide _______ different triplet combinations, more than enough to code for all the amino acids
43 = 64
If the code consisted of doublets and triplets, a signal denoting a _______ would once again be necessary. But a triplets-only code would require no symbol for a ______ if the mechanism for counting to three and distinguishing among successive triplets was very reliable
Indeed, groups of _____ nucleotides represent all 20 amino acids. Each nucleotide triplet is called a ______.
Each codon, designated by the bases defining its three nucleotides, specifies _____ amino acids. For example, GAA is a codon for ______ acid (Glu), and GUU is a codon for _____ (Val)
Why is it that geneticists usually present the code in RNA dialect of A, G, C, U and when speaking of genes, they substitute T for U to show the same code in the DNA dialect?
Because the code comes into play only during the translation part of gene expression, that is, during the decoding of messenger RNA to polypeptide
DNA is a _____ molecule with base pairs following one another down the interwined chains. _______, by contrast, have complicated three-dimensional structures
If proteins are unfolded and stretched out from their N terminus to C terminus, proteins have a _____-dimensional, ______ structure. What does this imply?
- implies a specific sequence of amino acids
If the info in a gene and its corresponding protein are colinear, the consecutive order of bases in the DNA from the beginning to the end of the gene would stipulate the consecutive order of ______ _____ from one end to the other of the outstretched protein
In the 1960s, Charles Yanofsky was the first to compare maps of mutations within a gene to the particular amino acid substitutions that resulted. (3-story) hint: trp- mutant in E. coli
- He began by generating a large number of trp- auxotrophic mutants in E. coli that carried mutations in the trpA gene for a subunit of the enzyme tryptophan synthetase
- He next made a fine structure recombinational map of these mutations
- Yanofsky then purified and determined the amino acid sequence of the mutant tryptophan synthetase subunits
What does the data of the figure illustrate?
- The order of mutations mapped within the DNA of the gene by recombination was indeed colinear with the positions of the amino acid substitutions occurring in the resulting mutant proteins
- *By carefully examining the results of his analysis, Yanofsky deduced key features of the relationship between nucleotides and amino acids, in addition to his confirmation of colinearity
Yanofsky observed that point mutations altering different nucleotide pairs may affect the _____ amino acids. In one example shown in the Fig, mutation 23 changed the glycine (Gly) at position 211 of the wild-type polypeptide chain to _______, while mutation 46 yielded _______ _______
- arginine (Arg)
- glutamic acid (Glu)
The point mutations here are all ______ mutations (explain). In both cases, Yanofsky also found that _______ could occur between the two mutations that changed the identity of the same amino acid; such ________ would produce a wildtype ______ ________ gene.
- missense mutations: change a codon for one amino acid into a codon that specifies a different amino acid
- tryptohan synthetase gene
Why is it that two mutations capable of recombination (in this case same codon affecting same amino acid) must be in different nearby nucleotides? What does this mean for the amount of nucleotides in a codon?
- Because the smallest unit of recombination is the base pair
- Result: a codon must contain more than one nucleotide
As fig. 8.3a illustrates, each of the point mutations in the tryptophan synthetase gene characterized by Yanofsky alters the identity of only _____ amino acid. This is also true of point mutations examined in many other genes, such as the human genes ________ and ________
Why is it that each nucleotide in a gene must influence the identity of only a single amino acid.
In contrast, if a nucleotide were part of more than one codon, a mutation in that nucleotide would affect?
- Because point mutations that change only a single nucleotide pair affect only a single amino acid in a polypeptide
- In contrast, that would affect more than one amino acid
- Crick and Brenner began their experiments with a particular proflavin-induced rIIB- mutation they called FC0
- They next treated this mutants strain with more proflavin to isolate an rIIB+ revertant (a)
- By recombining this revertant with wild-type bacteriophiage T4, Crick and Brenner were able to show that the revertant's chromosome actually contained two different rIIB- mutations (b)
- One was the original FC0 mutation; the other was the newly induced FC7
Either mutations (FC0/FC7 mutations) by itself yields a _______ phenotype, but their simultaneous occurrence in the same gene yielded an _____ phenotype
Crick and Brenner reasoned that if the first mutation was the _______ of a single base pair, represented by the symbol (__), then the counteracting mutation must be the _______ of a base pair, represented as (__)
The restoration of gene function by one mutation canceling another in the same gene
Crick and Brenner supposed not only that each codon is a trio of nucleotides, but that each gene has a single starting point which establishes a ______ _____ (define)
reading frame: the sequential partitioning of nucleotides into groups of three to generate the correct order of amino acids in the resulting polypeptide chain
Changes that alter the grouping of nucleotides into codons
Frameshift mutations shift the reading frame for all codons beyond the point of ______ or _______. What is the most likely result?
- insertion or deletion
- Polypeptide product won't function properly
If codons are read in order from a fixed starting point, a ________ can counterbalance an _______ to restore the reading frame.
- deletion (-)
- insertion (+)
Note that the gene would regain its wild-type activity only if the portion of the _________ encoded between the two mutations of opposite sign is not required for protein function (why)
- because in the double mutant, this region would have an improper amino acid sequence
Crick and Brenner realized that they could use + and - mutations in rIIB to test the hypothesis that codons were indeed _________ ________. If codons are composed of three nucleotides, then combining two different rIIB mutations of the same sign (++ or --) in the same gene should never lead to _______ ________.
- nucleotide triplets
- intragenic suppression (an rIIB+ phenotype)
Combinations of three + or three - mutations, however, should sometimes result in an _____ revertant. These predictions were exactly verified by the results.
Where does intragenic suppression occur?
- In the region between two frameshift mutations of opposite sign, a gene still dictates the appearance of amino acids
- *even if these amino acids are not the same as those appearing in the normal protein
If the frameshifted part of the gene instead encodes instructions to stop protein synthesis by introducing a triplet that does not correspond to any amino acid, then production of a functional polypeptide will not be possible (Why?)
The reason is that polyppeptide synthesis would stop before the compensating mutation could reestablish the correct reading frame
The fact that intragenic suppression occurs as often as it does suggests that the code inlcudes more than _____ _______ for some amino acids
Recall that there are 20 common amino acids but ______ different combos of three nucleotides. If each amino acid corresponded to only a single codon, there would be ________ possible triplets not encoding an amino acid.
These noncoding triplets would act as ____ signals and prevent further ________ synthesis
In this scenario, more than half of all frameshift mutations (44/64) would cause protein synthesis to stop at the _____ codon after the mutation, and the chances of extending the protein would ______ exponentially with each additional amino acid. As a result, _______ _______ would rarely occur
- first codon
- intragenic suppression
However, we have seen that frameshift mutations of one sign can be offset by mutations of the other sign. The distances between these mutations, estimated by recombination frequencies, are in some cases large enough to code for more than ____ amino acids, which would be possible only if (?)
- 50 amino acids
- Only if most of the 64 possible triplet codons specified amino acids
Thus, the data of Crick and Brener provide strong support for the idea that the genetic code is _______ (explain)
degenrative: two or more nucleotide triplets specify most of the 20 amino acids
In the 1950s, researchers exposed eukaryotic cells to amino acids tagged with radioactivity and observed that protein synthesis incorporating the radioactive amino acids into polypeptides take place in the _______, even though the genes for those polypeptides are sequestered in the cell ________
From this discovery, they deduced the existence of an intermediate molecule, made in the nucleus and capable of (?)
transporting DNA sequence info to the cytoplasm, where it can direct synthesis
Why is it that we can easily imagine the cellular machinery copying a strand of DNA into a complementary strand of RNA in a manner analogous to the DNA-DNA copying of DNA replication?
Because of RNA's potential for base pairing with a strand of DNA
Subsequent studies in eukaryotes using _______ ______, a base found only in RNA, showed that although the molecules are synthesized in the nucleus at least some of them migrate to the ______
- radioactive uracil
The main RNA molecule that migrate to the cytoplasm
mRNAs (messenger RNAs)
mRNAs arise in the nucleus from ________ of DNA sequence info and then move (after _______) to the cytopalsm, where they determine the proper order of amino acids during ______ synthesis
- protein synthesis
Knowledge of mRNA served the framework for two experimental breakthroughs that led to the deciphering of the genetic code. What were those breakthroughs?
- In the first, biochemists obtained cellular extracts that, with the addition mRNA, synthesized polypeptides in a test tube. They called these extracts "in vitro translational systems."
- The second breakthrough was the development of techniques enabling the synthesis of artificial mRNAs containing only a few codons of known composition
- *When added to in vitro translation systems these simple synthetic mRNAs directed the formation of very simple polyeptides
The appraoch Nirenberg and Philip used to see which small RNA caused the extrapment of which radioactively labeled amino acid (4-story)
- They added short synthetic mRNAs only three nucleotides in length to an in vitro translational system containing tRNAs attached to amino acids, where only one of the 20 amino acids was radioacitive
- They then poured through a filter a mixture of a synthetic mRNA and the translational system containing a tRNA-attached, radioactively labeled amino acid
- tRNAs carrying an amino acid normally go right through a filter
- If However, a tRNA carrying an amino acid binds to a ribosome, it will stick in the filter, because this larger complex of ribosome, amino acid carrying tRNA, and small mRNA cannot pass through
In studies using synthetic mRNAs, when investigators added the six-nucleotide-long 5' AAAUUU 3' to an in vitro tranlastional system, the product N Lys-Phe C emerged but no N Phe-Lys C appeared. Explain the results
- Because AAA is the codon for lysine and UUU is the codon for phenylalanine, this results means that the codon closest to the 5' end of the mRNA encoded the amino acid closest to the N terminus of the corresponding polypeptide
- *Similarly, the codon nearest the 3' end of the mRNA encoded the amino acid nearest the C terminus of the resulting polypeptide
To understand how the polarities of the macromolecules participating in gene expression relate to each other, remember that although the gene is a segment of a DNA double helix, only one of the two strands serves as a _______ for mRNA. This strand is known as the ______ _______
The other strand is the RNA-like strand (why?)
because it has the same polarity and sequence (written in the DNA dialect) as the RNA
Note that some scientists use the terms sense strand or coding strand as synonyms for the ____-_____ strand; in these alternative nomenclatures, the template would be the __________ strand or the ________ strand
- antisense strand or noncoding strand
Although most of the simple, repetitive RNAs synthesized by Khorana were very long and thus generated very long ________, a few did not. These RNAs had signals that ________ construction of the chain. The three triplets (name them) don't correspond to any of the ______ ________. Their purpose is to stop _________ when they appear in the frame
- UAA, UAG, UGA
- amino acids
Frameshift mutations: nucleotide insertions or deletions that alter the genetic instructions for polypeptide construction by changing the reading frame
- Missense mutations: change a codon for one amino acid to a codon for a different amino acid
- Nonsense mutations: change a codon for an amino acid to a stop codon
Most mutagens result in _____ nucleotide substitutions, and analyses of these substitutions should conform to the code. For example, Yanofsky found two trp- auxotrophoic mutations in the E. coli tryptophan synthetase gene that produced ____ _______ amino acids (________ or _______) at the ______ position (amino acid 211) in the polypeptide chain. According to the code, both of these mutations could have resulted from ______-base changes in the GGGA codon that normally inserts glycine (Gly) at position 211
- two different amino acids
- arginine or glutamic acid
Even more informative were the trp+ revertants of these mutations subsequently isolated by Yanofsky. As the fig illustrated, _____-base substitutions could also explain the amino acid changes in these revertants
Note that some of these substitutions restore Gly to position 211 of the polypeptide, while others place amino acids such as Ile, Thr, Ser, Ala, or Val at this site in the tryptophan synthetase molecule. The substitution of these other amino acids for Gly at position 211 in the polypetide chain is ________ (aka largely ________) with enzyme's function
Yanofsky obtained better evidence yet that cells use the genetic code in vivo by analyzing proflavin-induced ________ mutations of the tryptophan synthetase gene
Yanofsky first treated populations of E. coli with proflavin to produce _____ mutants. Subsequent treatemnt of these mutants with more proflavin generated some ____ revertants among the progeny. What is the most likely explanation?
- The most likely explanation for the revertants was that their tryptophan synthetase gene carried both a single-base-pair deletion and a single-base-pair insertion (+ -)
The universality of the code is an indication that it evolved very ______ in the history of life (explain)
- Once it emerged, it remained constant over billions of years, in part because evolving organisms would have little tolerance for change
An exception to the universality of the code would be some species of single-celled eukaryotic protozoans called _______. The codons UAA and UAG , which are ________ codons in most organisms, specify the amino acid glutamine; in other ciliates, UGA, the third ______ codon in most organisms, specifies cystein. ______ use the remaining ________ codons normally
- nonsense/stop codons
- nonsense/stop codon
- nonsense/stop codons
Yet another exception to the code is seen in certain prokaryotes which sometimes use the triplet UAG to specify insertion of the rare amino acid _______