Chapter 12: The Genetic Code and Transcription

The significance of transcription is enormous, for it is the initial step in the process of information flow within the cell. The idea that RNA is involved as an intermediate molecule in the process of information flow between DNA and protein was suggested by the following three observations:

(1) DNA is, for the most part, associated with chromosomes in the nucleus of the eukaryotic cell. However, protein synthesis occurs in association with ribosomes located outside the nucleus in the cytoplasm. Therefore, DNA does not appear to participate directly in protein synthesis. (2) RNA is synthesized in the nucleus of eukaryotic cells, where DNA is found, and is chemically similar to DNA (3) Following its synthesis, most messenger RNA migrates to the cytoplasm, where protein synthesis (translation) occurs. Collectively, these observations suggested that genetic information, stored in DNA, is transferred to an RNA intermediate, which directs the synthesis of proteins. These observations were supported by experiments.

When synthetic copolymer mRNAs were added to a cell-free system, the predicted number of amino acids incorporated into polypeptides was upheld. Several examples are shown in the table 12.3. When the data were combined with those on composition assignment and triplet binding, specific assignments were possible. One example of specific assignments made in this way illustrate the value of Khorana's approach. Consider the following experiments in concert with one another:

(1) The repeating trinucleotide sequence UUCUUCUUC.... can be read as three possible triplets-UUC, UCU, and CUU-depending on the initiation point. When placed in a cell-free translation system, three different polypeptide homopolymers-containing either phenylalanine, serine, or leucine-are produced. Thus, we know that each of the three triplets encodes one of the three amino acids, but we do not know which codes which. (2) On the other hand, the repeating dinucleotide sequence UCUCUCUC... produced the triplets UCU and CUC, and when used in an experiment, leads to the incorporation of leucine and serine into a polypeptide. Thus, the triplets UCU and CUC specify leucine and serine, but we still do not know which triplet specifies which amino acid. However, when considering both sets of results in concert, we can conclude that UCU, which is common to both experiments, must encode either leucine or serine but not phenylalanine. Thus either CUU or UUC encodes leucine or serine, while the other encodes phenylalanine From these and similar interpretations, Khorana reaffirmed the identity of triplets that had already been deciphered and filled in gaps left from other approaches. Of great interest, the use of two tetranucleotide sequences, GAUA and GUAA suggested that at least two triplets were termination codons. Khorana reached this conclusion because neither of these repeating sequences directed the incorporation of more than a few amino acids into a polypeptide, too few for him to detect. Of the possible triplets in poly-(GAUA), UAG was later shown to be a termination codon.

The genetic coding dictionary reveals the function of _______ triplets. The various techniques used to decipher the genetic code have yielded a dictionary of ____ triplet codons assigned to amino acids.

64 61 The remaining three triplets are termination signals and do not specify any amino acid

The complete coding dictionary reveals that of the _____ possible triplet codons, ____ encode the ______ amino acids found in proteins, while _____ triplets terminate translation

64 61 20 3

An important posttranscriptional of eukaryotic RNA transcripts destined to become mRNAs occurs at the 5'-end of these molecule, where a __________________ is added.

7-methylguanosine (m^7G) cap

In the early 1960s, Sydney Brenner argued on theoretical grounds that the genetic code had to be a triplet since three-letter words represent the minimal use of four letters to specify 20 amino acids. Explain this.

A code of four nucleotides, taken two at a time, for example, provides only 16 unique code words (4^2). A triplet code yields 64 words (4^3)-clearly more than the 20 needed-and is much simpler than a four-letter code (4^4) which specifies 256 words

The genetic code is degenerate or redundant . What does this mean?

A given amino acid can be specified by more than one triplet codon. This is the case for 18 of the 20 amino acids.

Only one codon, ____________, codes for methionine, and it is sometimes called the initiator codon.

AUG However, when AUG appears internally in mRNA, rather than atan initiating position, unformylated methionine is inserted into the polypeptide chain

RNA polymerase from E.coli has been extensively characterized and shown to consist of subunits designated α (alpha), β (beta), β' (beta prime), and ω (omega). The core enzyme contains the subunits _________ (two copies), ________, _______, and ______. A slightly more complex form of the enzyme, the ____________ contains an additional subunit called the _______________.

Alpha (α)(two copies), Beta (β), Beta prime (β'), and Omega (ω). holoenzyme sigma (σ) factor

Table 12.6 RNA polymerases in eukaryotes

As noted earlier, eukaryotic RNA polymerase exists in three distinct forms. Each eukaryotic RNA polymerase is larger and more complex than the single form of RNA polymerase found in bacteria. For example, yeast and human RNA polymerase II enzymes consist of 12 subunits. While the three forms of the enzyme share certain protein subunits, each nevertheless transcribes different types of genes.

Yet another innovative technique used to decipher the genetic code was developed in the early 1960s by Har Gobind Khorana, who chemically synthesized long RNA molecules consisting of short sequences repeated many times. First, he created shorter sequences (F.E., di-, tri-, and tetra nucleotides), which were then replicated many times and finally joined enzymatically to form the long polynucleotides, referred to as copolymers. Figure 12.6

As shown in the figure, a dinucleotide made in this way is converted to an mRNA with two repeating triplets. A trinucleotide is converted to an mRNA with three potential triplets, depending on the point at which initiation occurs, and a tetranucleotide creates four repeating triplets.

Reaction summarizing the synthesis of RNA on a DNA template

As this equation reveals, nucleoside triphosphates (NTPs) are substrates for the enzyme, which catalyzes the polymerization of nucleoside monophosphates (NMPs) or nucleotides, into a polynucleotide chain (NMP)n. Nucleotides are linked during synthesis by 3' to 5' phosphodiester bonds. The energy created by cleaving the triphosphate precursor into the monophosphate form drives the reaction, and inorganic pyrophosphates (PPi) are produced (Ribonucleotides are added not deoxyribonucleotides)

A second equation summarizes the sequential addition of each ribonucleotide as the process of transcription progresses

As this equation shows, each step of transcription involves the addition of one ribonucleotide (NMP) to the growing polyribonucleotide chain (NMP)n+1 using a nucleoside triphosphate (NTP) as the precursor.

The various aspects of the genetic code discussed thus far yield a fairly complete picture, suggesting that it is triplet in nature, degenerate, unambiguous, and commaless, and that it contains punctuation start and stop signals. That these features are correct was confirmed by analysis of the RNA-containing ____________________ by Walter Fiers and his coworkers

Bacteriophage MS2

The _____ and ______ polypeptides provide the catalytic basis and active site for transcription, while the _______ plays a regulatory function in the initiation of RNA transcription.

Beta (β) and Beta prime (β') σ factor

Another observation in the pattern of codon sequences and their corresponding amino acids is an ordered genetic code. What does this mean?

Chemically similar amino acids often share one or two "middle" bases in the different triplets encoding them. For example, either U or C is often present in the second position of triplets that specify hydrophobic amino acids. The chemical properties of amino acids will be discussed more in chapter 13 but the end result of an "ordered" genetic code is that it buffers the potential effect of mutation on protein function. While many mutations of the second base of triplet codons result in a change of one amino acid to another, the change is often to an amino acid with similar chemical properties. In such cases, protein function may not be noticeably altered

T/F The 5' cap is added long after synthesis of the initial RNA transcript has begun

F the cap is added shortly after synthesis of the initial RNA transcript has begun

Experimental evidence supporting the triplet nature of the code was subsequently derived from research by Francis Crick and his colleagues. Using phage T4, they studied frameshift mutations. How were they able to apply frameshift mutations to the genetic code and what were the results?

Frameshift mutations result from the addition or deletion of one or more nucleotides within a gene and subsequently the mRNA transcribed by it. The gain or loss of letters shifts the frame of reading during translation. Crick and his colleagues found that the gain or loss of one or two nucleotides caused a frameshift mutation, but when three nucleotides were involved, the frame of reading was reestablished. This would not occur if the code was anything other than triplet. This work also suggested that most triplet codes are not blank, but rather encode amino acids, supporting the concept of a degenerate code.

Explanation of the poly-A tail mRNA processing

Further insights into the processing of RNA transcripts during the maturation of mRNA came from the discovery that mRNAs contain, at their 3'-end, a stretch of as many as 250 adenylic acid residues. As discussed earlier in the context of eukaryotic transcription termination, the transcript is cleaved roughly 10 to 35 ribonucleotides after the highly conserved AAUAAA polyadenylation signal sequence. An enzyme known as poly-A polymerase then catalyzes the addition of a poly-A tail to the free 3'-OH group at the end of the transcript. Poly-A tails are found at the 3'-end of almost all mRNAs studied in a variety of eukaryotic organisms. The exceptions in eukaryotes seem to be mRNAs that encode histone proteins. While the AAUAAA signal sequence is not found on all eukaryotic mRNAs, it appears to be essential to those that have it. If the sequence is changed as a result of a mutation, those transcripts that would normally have it cannot add the poly-A tail. In the absence of this tail, these RNA transcripts are rapidly degraded by nucleases.

Figure 12.1 Flowchart of how genetic information encoded in DNA produced proteins (simplified)

In the first step in gene expression, information on one of the two strands of DNA (the template strand) is copied into an RNA complement through transcription. Once synthesized, this DNA acts as a "messenger" molecule bearing the coded information-hence its name, messenger RNA (mRNA). The mRNAs then associate with ribosomes, where decoding into proteins takes place.

The use of Homopolymers by Nirenberg and Matthaei in their initial experiments of deciphering the genetic code

In their initial experiments, Nirenberg and Matthaei synthesized RNA homopolymers, each with only one type of ribonucleotide. Therefore, the mRNA added to the in vitro system was UUUU..., AAAA..., CCCC..., or GGGG.. They tested each mRNA and were able to determine which, if any, amino acids were incorporated into newly synthesized proteins. To do this, the researchers labeled 1 of the 20 amino acids added to the in vitro system and conducted a series of experiments, each with a different radioactively labeled amino acid. For example, in experiments using 14C-phenylalanine, Nirenberg and Matthaei concluded that the message poly U (polyuridylic acid, UUU...) directed the incorporation of only phenylalanine into the homopolymer polyphenylalanine. Assuming the validity of a triplet code, they determined the first specific codon assignment-UUU codes for phenylalanine. -Using similar experiments, they quickly found that AAA code for lysine and CCC codes for proline. Poly G was not an adequate template, probably because the molecule folds back upon itself. Thus, the assignment for GGG has to await other approaches Note that the specific triplet codon assignments were possible only because homopolymers were used. This method yields only the composition of triplets, but since three identical letters can have only one possible sequence (UUU for example) the actual codons were identified.

Experiments using RNA heteropolymers done by Nirenberg and Matthaei involves the use of: (includes some background as well)

In this type of experiment, two or more different ribonucleoside diphosphates are added in combination to form the synthetic mRNA. The researchers reasoned that if they knew the relative proportion of each type of ribonucleoside diphosphate, they could predict the frequency of any particular triplet codon occuring in the synthetic mRNA. If they then added the mRNA to the cell free system and ascertained the percentage of any particular amino acid present in the new protein, they could analyze the results and predict the composition (but not the specific sequence) of triplets specifying particular amino acids.

Figure 12.7 The coding dictionary

Most evident is that the code is degenerate that is almost all amino acids are specified by two, three, or four different codons. Three amino acids (serine, arginine, and leucine) are each encoded by six different codons. Only tryptophan and methionine are encoded by single codons. The pattern of degeneracy is also evident. Most often, in a set of codons specifying the same amino acid, the first two letters are the same, with only the third differing. Crick discerned a pattern in the degeneracy at the third position, and he postulated the wobble hypothesis.

There are two different types of transcription termination mechanisms in bacteria, both of which are dependent on the formation of a hairpin structure in the RNA being transcribed. What are these mechanisms and explain them.

Most transcripts in E.coli are terminated by intrinsic termination. In intrinsic termination, a hairpin structure encoded by the termination sequence causes RNA polymerase to stall. Immediately after the hairpin is a string of uracil residues. The U bases of the transcript have a relatively weak interaction with the A bases on the template strand of the DNA because there are only two hydrogen bonds per base pair. This leads to the dissociation of RNA polymerase and the transcript is released. Other bacterial transcripts are terminated by rho-dependent termination, which involves a termination factor called rho (ρ). Rho is a large hexameric protein with RNA helicase activity-it can dissociate RNA hairpins and DNA-RNA interactions. Rho binds to a specific sequence on the transcript and move in the 3' direction chasing after RNA polymerase. When RNA polymerase reaches the hairpin structure encoded by the termination sequence, it pauses and rho catches up. Rho moves through the hairpin and then causes dissociation of RNA polymerase by breaking the hydrogen bonds between the DNA template and the transcript.

The genetic code is nonoverlapping. What does this mean?

Once translation commences, any single ribonucleotide at a specific location within the mRNA is part of only one triplet.

Where are promoters located?

Promoter regions are located in the region upstream (5') from the point of initial transcription of a gene. It is believed that the enzyme "explores" a length of DNA until it recognizes the promoter region and binds to about 60 nucleotide pairs of the helix, 40 of which are upstream from the point of initial transcription.

Transcription synthesizes _______ on a ________ template

RNA DNA

RNA Polymerase ____ and _____ transcribe transfer RNAs (tRNAs) and ribosomal RNAs (rRNAs), which are needed in essentially all cells at all times for the basic process of protein synthesis.

RNA Polymerase I and III (RNAP I and RNAP III)

Once mRNA was discovered, it was clear that even though genetic information is stored in DNA, the code that is translated into proteins resides in _______________

RNA or mRNA more specifically

To establish that RNA can be synthesized on a DNA template, it was necessary to demonstrate that there is an enzyme capable of directing this synthesis. By 1959, several investigators including Samuel Weiss had independently isolated such a molecule from rat liver. This molecule is called ________________

RNA polymerase

RNA polymerase ___, which transcribes protein-coding genes, is highly regulated. Protein-coding genes are often expressed at different times, in response to different signals, and in different cell types. Thus, RNAP ___ activity is tightly regulated on a gene-by-gene basis.

RNA polymerase II RNAP II

Figure 12.4 Experimental Approach using RNA heteropolymers

Suppose that A and C are added in a ratio of 1A:5C. The insertion of a ribonucleotide at any position along the RNA molecule during its synthesis is determined by the ratio of A:C (due to the nature of polynucleotide phosphorylase). Therefore, there is a 1/6 chance for an A and a 5/6 chance for a C to occupy each position. On this basis, we can calculate the frequency of any given triplet appearing in the message. -For AAA, the frequence is (1/6)^3, or about 0.4%. For AAC, ACA, and CAA, the frequencies are identical-that is, (1/6)^2(5/6), or about 2.3 percent for each triplet. Together, all three 2A:1C triplets account for 6.9 percent of the total three-letter sequences. In the same way, each of three 1A:2C triplets accounts for (1/6)(5/6)^2, or 11.6 percent (or a total 34.8 percent); CCC is represented by (5/6)^3, or 57.9 percent of the triplets. By examining the percentages of any given amino acid incorporated into the protein synthesized under the direction of this message, we can propose probably base compositions for each amino acid. Since proline appears 69 percent of the time, we could propose that proline is encoded by CCC (57.9 percent) and one triplet of 2C:1A (11.6 percent). Histidine, at 14 percent, is probably coded by one 2C:1A (11.6 percent) and one 1C:2A (2.3 percent). Threonine, at 12 percent, is likely coded by only one 2C:1A. Asparagine and glutamine each appear to be coded by one of the 1C:2A triplets, and lysine appears to be coded by AAA. Using as many as all four ribonucleotides to construct the mRNA, the researchers conducted many similar experiments. Although determining the composition of the triplet code words for all 20 amino acids represented a significant breakthrough, the specific sequences of triplets were still unknown-other approaches were needed.

T/F After a few ribonucleotides have been added to the growing RNA chain, the σ factor dissociates from the holoenzyme and elongation proceeds under the direction of the core enzyme.

T

T/F Although the base sequence of DNA in bacteria is transcribed into an mRNA that is immediately and directly translated into the amino acid sequence as dictated by the genetic code, eukaryotic mRNAs require significant alteration before they are transported to the cytoplasm and translated

T

T/F Although there is but a single form of the core enzyme in E.coli, there are several different σ factors, creating variations of the polymerase holoenzyme. On the other hand, eukaryotes display three distinct forms of RNA polymerase, each consisting of a greater number of polypeptide subunits than in bacteria.

T

T/F Chemically, the cap is a guanosine residue with a methyl group (CH3) at position 7 of the base. The cap is also distinguished by a unique 5' to 5' triphosphate bridge that connects it to the initial ribonucleotide of the RNA

T

T/F In addition, the poly-A tail is important for export of the mRNA from the nucleus to the cytoplasm and for translation of the mRNA

T

T/F In bacteria, either the formyl group is removed from the initial methionine upon the completion of protein synthesis or the entire formylmethionine residue is removed.

T

T/F In bacteria, transcription termination is often dependent upon the formation of a hairpin secondary structure in the transcript. However, eukaryotic transcription termination is more complex. Transcriptional termination for protein-coding genes involves sequence-specific cleavage of the transcript, which then leads to eventual dissociation of RNA polymerase from the DNA template.

T

T/F In contrast to RNA polymerase, polynucleotide phosphorylase does not require a DNA template.

T

T/F In eukaryotes, methionine (AUG) is also the initial amino acid during polypeptide synthesis. However, it is not formylated

T

T/F In some eukaryotic genes, a cis-acting element within the core promoter is the Goldberg-Hogness box or TATA box. Located about 30 nucleotide pairs upstream (-30) from the start point of transcription, TATA boxes share a consensus sequence TATA^A/T^AAR where R indicates any purine nucleotide. The sequence and function of TATA boxes are analogous (comparable in certain respects, typically in a way which makes clearer the nature of the things compared) to those found in the -10 promoter region of bacterial genes. However, recall that in bacteria, RNA polymerase binds directly to the -10 promoter region. As we will see soon, the same is not the case in eukaryotes

T

T/F Much of our knowledge of transcription has been derived from studies of bacteria. The general aspects of the mechanics of these processes are mostly similar in eukaryotes, but there are several notable differences.

T

T/F The activity of RNAP II is dependent on both the cis-acting regulatory elements of the gene and a number of trans-acting transcription factors that bind to these DNA elements.

T

T/F The genetic code is nearly universal

T

T/F The linear sequence of deoxyribonucleotides making up DNA ultimately dictates the components constituting proteins, the end product of most genes.

T

T/F The major form of the σ factor in bacteria is designated as σ^70 based on its molecular weight of 70 kilodaltons (kDa). The promoters of most bacterial genes are recognized by this form; however, several alternative σ factors (eg., σ^32, σ^54, etc.) are called upon to regulate other genes.

T

T/F in contrast to the in vitro experiments discussed earlier, initiation of protein synthesis in vivo is a highly specific process

T

T/F unlike DNA polymerase, no primer is required to initiate synthesis.

T

T/F Introns are common in humans; roughly 94 percent of human protein-coding genes contain introns with an average of nine exons and eight introns per gene.

T Although the vast majority of mammalian genes examined thus far contain introns, there are several exceptions. Noteable, the genes coding for histones and for interferon, a signaling protein of the immune system, appear to contain no introns.

T/F Unlike in bacteria, there is no specific sequence that signals for termination of transcription.

T RNAP II often continues transcription well beyond what will be the eventual 3'-end of the mature mRNA. Once transcription has incorporated a specific sequence AAUAAA, known as the polyadenylation signal sequence, the transcript is enzymatically cleaved roughly 10 to 35 bases downstream in the 3' direction. Cleavage of the transcript destabilizes RNAP II, and both DNA and RNA are released from the enzyme as transcription is terminated. This complete the cycle that constitutes transcription.

T/F The degree of RNA polymerase binding to different promoters varies greatly, causing variable gene expression. Currently, this is attributed to sequence variation in the promoters.

T In bacteria, both strong promoters and weak promoters have been discovered, causing a variation in time of initiation from once every 1 to 2 seconds to as little as 10 to 20 minutes. Mutations in promoter sequences may severely reduce the initiation of gene expression.

T/F In the late 1950s, before it became clear that mRNA is the intermediate that transfers genetic information from DNA to proteins, researchers thought that DNA itself might directly encode proteins during their synthesis.

T The initial thinking was that information in DNA was transferred in the nucleus to the RNA of the ribosome, which served as the template for protein synthesis in the cytoplasm. This concept soon became untenable as accumulating evidence indicated the existence of an unstable intermediate template.

The general transcription factors involved with human RNAP II binding are well characterized and are designated _____, _______, and so on.

TFIIA, TFIIB, and so on.

_______ binds directly to the TATA-box sequence.

TFIID

The probability of the insertion of a specific ribonucleotide by polynucleotide phosphorylase is proportional to:

The availability of that molecule, relative to other available ribonucleotides.This point is absolutely critical to understanding the work of Nirenberg and others in the ensuing discussion. Together, the cell free system and the availability of synthetic mRNAs provided a means of deciphering the ribonucleotide composition of various triplets encoding specific amino acids.

What is the caps function?

The cap stabilizes the mRNA by protecting the 5'-end of the molecule from nuclease attack. Subsequently, the cap facilitates the transport of mature mRNAs from the nucleus into the cytoplasm and is required for the initiation of translation of the mRNA into protein.

Table 12.5 Exception to the universal genetic code

The change in coding capacity involves only a shift in recognition of the third, or wobble, position. For example, AUA specifies isoleucine in the cytoplasm and methionine in the mitochondrion, but in the cytoplasm, methionine is specified by AUG. Similarly, UGA calls for termination in the cytoplasm, but it specifies tryptophan in the mitochondrion; in the cytoplasm, tryptophan is specified by UGG. It has been suggested that such changes in codon recognition may represent an evolutionary trend toward reducing the number of tRNAs needed in the mitochondria.

Figure 12.5 Illustration of the behavior if components during the triplet binding assay.

The triplet sequence in the experiment acts like a codon of mRNA, attracting a transfer RNA molecule containing the complementary sequence and carrying a specific amino acid. The triplet sequence in tRNA that is complementary to a codon of mRNA is called an anticodon. Although it was not feasible to chemically synthesize long stretches of RNA, triplets of known sequence could be synthesized in the laboratory to serve as templates. All that was needed was a method to determine which tRNA-amino acid was bound to the triplet RNA-ribosome complex. The test system Nirenberg and Leder devised was quite simple. The amino acid to be tested was made radioactive, and a charged tRNA was produced. Because codon compositions were known (from the RNA homo/hetero polymer experiments), researchers could narrow the range of amino acids that should be tested for each specific triplet. The radioactively charged tRNA, the RNA triplet, and ribosomes were incubated together and then passed through a nitrocellulose filter, which retains the larger ribosomes but not the other smaller components, such as unbound charged tRNA. If radioactivity is not retained on the filter, an incorrect amino acid has been tested. But if radioactivity remains on the filter, it is retained because the charged tRNA has bound to the triplet associated with the ribosome. When this occurs, a specific codon assignment can be made to a amino acid.

What are Consensus sequences of DNA?

These sequences are similar (homologous) in different genes of the same organism or in one or more genes of related organisms. Their conservation throughout evolution attests to the critical nature of their role in biological processes.

Table 12.2 Amino acid Assignments to Specific Trinucleotides derived from the Triplet Binding Assay

These specific assignments of triplets to amino acids led to two major conclusions. First, the genetic code is degenerate; that is, one amino acid can be specified by more than one triplet. Second, the code is unambiguous. That is, a single triplet specifies only one amino acid. The triplet binding technique was a major innovation in deciphering the genetic code

Figure 12.3 The reaction catalyzed by the enzyme polynucleotide phosphorylase

This enzyme, isolated from bacteria, catalyzes the reaction shown. The enzyme functions metabolically in bacterial cells to degrade RNA. However, in vitro, with high concentrations of ribonucleotide diphosphates, the reaction can be "forced" in the opposite direction to synthesize RNA, as shown. (shifting the reaction using equilibrium properties)

The wobble hypothesis explained

This hypothesis first predicted that the initial two ribonucleotides of triplet codes are more critical than the third in attracting the correct tRNA during translation. He postulated that hydrogen bonding at the third position of the codon-anticodon interaction is less constrained and need not adhere to the established base-pairing rules. The wobble hypothesis thus proposes a more flexible set of base-pairing rules at the third position of the codon. This relaxed base-pairing requirement, or "wobble," allows the anticodon of a single form of tRNA to pair with more than one triplet in mRNA. Consistent with the wobble hypothesis and degeneracy, U at the first position (the 5'-end) of the tRNA anticodon may pair with A or G at the third position (the 3'-end)of the mRNA codon, and G may likewise pair with U or C. Inosine (I), one of the modified bases found in tRNA, may pair with C, U, or A. Applying these wobble rules, a minimum of about 30 different tRNA species is necessary to accommodate the 61 triplets specifying an amino acid. -Current estimates are that 30 to 40 tRNA species are present in bacteria and up to 50 tRNA species exist in animal and plant cells.

The genetic code is unambiguous. What does this mean?

This means that each triplet specifies only a single amino acid

In 1964, Nirenberg and Leder developed the ________________________________, which led to specific assignments of triplets. The technique took advantage of the observation that ribosomes, when presented in vitro with an RNA sequence as short as three nucleotides, will bind to it and form a complex similar to that found in vivo (in a living organism).

Triplet binding assay

Three triplets _____________, _______________, and _________________ serve as termination codons, punctuation signals that do not code for any amino acid. They are not recognized by a tRNA molecule, and translation terminates when they are encountered.

UAG, UAA, UGA Mutations that produce any of the three triplets internally in a gene will also result in termination. Consequently, only a partial polypeptide has been synthesized when it is prematurely released from the ribosome. When such a change occurs in the DNA, it is called a nonsense mutation.

The genetic code is nearly universal. What does this mean?

With only minor exceptions, almost all viruses, bacteria, archaea, and eukaryotes use a single coding dictionary.

In molecular genetics, then, cis-elements are ___________ parts of the same DNA molecule. This is in contrast to ___________________, molecules that bind to these DNA elements to influence gene expression

adjacent trans-acting factors

In 1961, Nirenberg and Matthaei deciphered the first specific coding sequences, which served as a cornerstone for the complete analysis of the genetic code. Their success, as well as that of other who made important contributions to breaking the code, was dependent on the use of two experimental tools- they are...

an in vitro (cell free) protein-synthesizing system and an enzyme, polynucleotide phosphorylase, which enabled the production of synthetic mRNAs. These mRNAs are templates for polypeptide synthesis in the cell-free system

Each triplet codon in the mRNA is, in turn, complementary to the _______________ region of its corresponding tRNA as the amino acid is correctly inserted into the polypeptide chain during translation.

anticodon

Why are the general transcription factors essential

because RNAP II cannot bind directly to eukaryotic core-promoter sites and initiate transcription without their presence.

Why is the complementary strand of the template strand called the coding strand in transcription

because it and the RNA molecule transcribed from the template strand have the same 5' to 3' nucleotide sequence, but with uridine (U) substituted for thymidine (T) in RNA.

Subsequent ribonucleotide complements are inserted and linked by phosphodiester bonds as RNA polymerization proceeds following initiation. This process of ____________ continues in a _____________ extension, creating a temporary DNA/RNA duplex whose chains run antiparallel to one another.

chain elongation 5'-3'

Initiation of transcription of eukaryotic genes requires that compact chromatin fiber, characterized by nucleosome coiling, be uncoiled to make the DNA helix accessible to RNA polymerase and other regulatory proteins. This transition, referred to as __________, reflects the dynamics involved in the conformational change that occurs as the DNA helix is opened.

chromatin remodeling

Two such consensus sequences have been found in bacterial promoters. One TATAAT, is located 10 nucleotides upstream from the site of initial transcription (the -10 region, or Pribnow box). The other, TTGACA, is located 35 nucleotides upstream (the -35 region). Mutations in either region diminish transcription, often severely. Sequences such as these are said to be _____________________

cis-acting DNA elements. Use of the term cis is drawn from organic chemistry nomenclature, meaning "next to" or on the same side as, in contrast to being "across from," or trans, to other functional groups.

In bacteria, groups of genes whose products function together are often clustered along the chromosome. In many such cases, they are contiguous (sharing a common border, touching), and all but the last gene lack the termination sequence. The result is that during transcription, a large mRNA is produced that encodes more than one protein. Since genes in bacteria are sometimes called _________, the RNA is called a ______________________. The products of genes transcribed in this fashion are usually all needed by the cell at the same time, so this is an efficient way to transcribe and subsequently translate the needed genetic information.

cistrons polycistronic mRNA polycistronic mRNAs are rare in eukaryotes.

MS2 is a bacteriophage that infects E.coli. Its nucleic acid (RNA) contains only about 3500 ribonucleotides, making up only four genes, specifying a coat protein, an RNA replicase, a lysis protein, and a maturation protein. The small genome and a few gene products enabled Fiers and his colleagues to sequence the genes and their products. When the chemical constitution of these genes and their encoded proteins were compared, they were found to exhibit ______________.

colinearity. That is, based on the coding dictionary, the linear sequence of triplet codons corresponds precisely with the linear sequence of amino acids in each protein. Punctuation was also confirmed. For example, in the coat protein gene, the codon for the first amino acids is AUG, the common initiator codon. The codon for the last amino acid is followed by two consecutive termination codons, UAA and UAG. The analysis clearly showed that the genetic code in this virus was identical to that established experimentally in bacterial systems.

T/F The use of RNA homopolymers and mixed heteropolymers in a cell-free system allowed the determination of the ____________________, but not the ________________, of triplet codons designating specific amino acids.

composition but not the sequence

At least four different types of cis-acting DNA elements regulate the initiation of transcription by RNAP II. The first of these, the ____________, includes the transcription start site. It determines where RNAP II binds to the DNA and where it begins transcribing the DNA into RNA. Another promoter element, called a ___________________, is located upstream of the start site and helps modulate the level of transcription. The last two types of cis-acting elements, called _________ nad _________, influence the efficiency or the rate of transcription initiation by RNAP II from the core-promoter element

core promoter proximal-promoter element enhancers silencers

Studies done by Nirenberg, Matthaei, and others ____________ the genetic code

deciphered

Each σ factor in bacteria recognizes _________ promoter sequences, which in turn provides specificity to the initiation of transcription

different

Transcriptional activators and repressors bind to _______ and ________ elements and regulate transcription initiation by aiding or preventing the assembly of pre-initiation complexes and the release of RNAP II from pre-initiation into full transcription elongation.

enhancer silencer

In addition to promoters, eukaryotic genes often have other cis-acting control units called _______ and ______ which greatly influence transcriptional activity.

enhancers silencers

T/F a primer is required for initiation during transcription

f

Initiation and regulation of transcription entail a more extensive interaction between cis-acting DNA sequences and trans-acting protein factors. For example, while bacterial RNA polymerase requires only a σ factor to bind the promoter and initiate transcription, in eukaryotes, several ___________________ are required to bind the promoter, recruit RNA polymerase, and initiate transcription.

general transcription factors (GTFs)

There are two broad categories of transcription factors: the _______________ that are absolutely required for all RNAP II-mediated transcription, and the ____________ and ____________ that influence the efficiency or the rate of RNAP II transcription initiation

general transcription factors (GTFs) Transcriptional activators and Transcriptional repressors

Eventually, the enzyme traverses the entire gene until it encounters a specific nucleotide sequence that acts as a termination signal. An interesting aspect of termination in bacteria is that the termination sequence alluded to above is actually transcribed into RNA. The unique sequence of nucleotides in this termination region causes the newly formed transcript to fold back on itself, forming what is called a __________________, held together by hydrogen bonds.

hairpin secondary structure

Once it has recognized and bound to the promoter, RNA polymerase catalyzes ___________, the insertion of the first 5'-ribonucleoside triphosphate.

initiation

The coding regions of eukaryotic genes are interrupted by intervening sequences called ________

introns

The primary mRNA transcript, or pre-mRNA, is often longer than the mature mRNA in eukaryotes. An explanation emerged in 1977 when Sharp and Roberts independently published direct evidence that the genes of animal viruses contain internal (also referred to as intervening or intragenic) nucleotide sequences that do not encode for amino acids in the final protein product. These noncoding internal sequences are also present in pre-mRNAs, but they are removed during RNA processing to produce the mature mRNA, which is then translated. Such nucleotide sequences-ones that intervene between sequences that code for amino acids-are called _______ .Sequences that are retained in the mature mRNA and expressed are called ________. The process of removing introns from a pre-mRNA and joining together exons is called ___________

introns exons RNA splicing

Transcription results in an ___________ molecule complementary to the gene sequence of ____________ of the double helix's two strands.

mRNA one

In 1961, Francois Jacob and Jacques Monod postulated the existence of _______________________

messenger RNA (mRNA)

in bacteria, the initial amino acid inserted into all polypeptide chains is a modified form of _____________________-N-formylmethionine (fMET).

methionine

Transcription in eukaryotes occurs within the _______. Thus unlike the bacterial process, in eukaryotes the RNA transcript is not free to associate with ribosomes (which are located in the cytoplasm) prior to the completion of transcription. For the mRNA to be translated, it must move out of the ________ into the ________

nucleus nucleus cytoplasm

Notes stop here on page 234. Should i read and take notes on 234-237? see if he talks about any of it in lecture

ok

The next group of cards is going to talk about an experiment that was used to decipher the genetic code by work done by Nirenberg and colleagues, all the cards pertain to the experiment until new shit comes up that doesnt involve learning experiments

ok

The genetic code contains ________ "start" and _______ "stop" signals (or codons), triplets that initiate and terminate translation

one three

The ______________________ binds to poly-A tails and prevents nucleases from degrading the 3'-end of the mRNA.

poly-A binding protein

In 1961, mRNA had yet to be isolated. However, use of the enzyme ______________________ allowed the artificial synthesis of RNA templates, which could be added to the cell-free system.

polynucleotide phosphorylase

once initial binding of TFIID to DNA occurs, the other general transcription factors, along with RNAP II, bind sequentially to TFIID, forming an extensive ______________________

pre-initiation complex

By 1970, evidence showed that eukaryotic mRNA is transcribed initially as a precursor molecule much larger than that which is translated into protein. It was proposed that this primary transcript of a gene (a __________) must be processed in the nucleus before it appear is the cytoplasm as a _______________ molecule to be translated.

pre-mRNA mature mRNA

In eukaryotes, the initial (or primary) transcripts of protein-coding mRNAs, called __________, undergo complex alterations, generally referred to as "processing," to produce a mature mRNA. Processing often involves the addition of a _____-cap and a ____-tail, and the removal of intervening sequences that are not a part of the mature mRNA.

pre-mRNAs 5'-cap 3'-tail

In bacteria, the site if template binding is established when the RNA polymerase σ factor recognizes specific DNA sequences called ____________

promoters

In contrast to RNA polymerase, polynucleotide phosphorylase does not require a DNA template. As a result, each addition of a ribonucleotide is random, based on the.....

relative concentration of the four ribonucleoside diphosphates added to the reaction mixtures.

RNA polymerase has the same general substrate requirements as does DNA polymerase, the major exception being that the substrate nucleotides contain the _______________ rather than the _____________ form of the sugar

ribose deoxyribose

Transcription results in the synthesis of a _________ stranded RNA molecule complementary to a region along ________ strand of the DNA double helix

single stranded one

T/F Most triplet code words direct the incorporation of a specific amino acid into a protein as it is synthesized

t

T/F No internal punctuation (such as commas) is used in the code. Thus, the code is said to be commaless. Once translation of mRNA begins, the codons are read one after the other, with no breaks between them.

t

The initial step in transcription is _____________

template binding

When discussing transcription, the DNA strand that serves as a template for RNA polymerase is denoted as the ______________ and the complementary DNA strand is called the ___________

template strand coding strand

The genetic code is written in linear form, using the ribonucleotide bases that compose mRNA molecules as "letters". The ribonucleotide sequence is derived from...

the complementary nucleotide bases in DNA.

In the cell-free system, amino acids are incorporated into polypeptide chains. What must this in vitro mixture contain

the essential factors for protein synthesis in the cell: ribosomes, tRNAs, amino acids, and other molecules essential to translation (see chapter 13). In order to follow (or trace) protein synthesis, one or more of the amino acids must be radioactive. Finally, an mRNA must be added, which serves as the template that will be translated.

Use of Triplet binding assay and of repeating copolymers allowed the determination of

the specific sequences of triplet codons designating specific amino acids.

The genetic code is written in units of ________ letters-ribonucleotides present in mRNA that reflect the stored information in genes.

three

Each "word" within the mRNA consists of __________ ribonucleotide letters, thus representing a ________ code. With several exceptions, each group of __________ ribonucleotides, called a codon, specifies one ___________________

three triplet three amino acid

Transcription in eukaryotes occurs under the direction of ___________________ of RNA polymerase, rather than the _______ of RNA polymerase seen in bacteria.

three separate forms single form

Even while the genetic code was being studied, it was quite clear that proteins were the end products of many genes. Thus, while some geneticists attempted to elucidate the code, other research efforts focused on the nature of genetic expression. The central question was how DNA, a nucleic acid, could specify a protein composed of amino acids. The complex multistep process begins with the transfer of genetic information stored in DNA to RNA. The process by which RNA molecules are synthesized on a DNA template is called ______________________

transcription

Complementing the cis-acting regulatory sequences are various trans-acting factors that facilitate RNAP II binding and, therefore, the initiation of transcription. These proteins are referred to as _______________

transcription factors

Once the RNA polymerase binds to the promoter region, the helix is denatured or unwound locally, making the DNA template accessible to the action of the enzyme. The point at which transcription actually begins is called the ___________________, often indicated as position +1

transcription start site

The promoters of bacteria are recognized by the _______________ in bacteria

σ factor