Genetics Ch. 13

Like DNA replication, the processes of transcription can be subdivided into the stages of initiation, elongation, and termination. Also, like DNA replication, transcription relies on base pairing affinities between complementary nucleotides.

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Look at chart printed for results of the previous experiment

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RNA editing alters the ribonucleotide sequence of an mRNA molecule prior to its translation

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RNA polymerase 2 must open up the DNA Helix and locally separate (denature) the two strands so that the template strand may pass through its active site during RNA synthesis.

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The Sigma factor plays a regulatory function in the initiation of RNA transcription

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The cap stabilizes the mRNA by protecting the 5' end of the molecule from nucleus attack the caps facilitates the transport of mature mRNA's from the nucleus into the cytoplasm is required for the initiation of translation of the mRNA into protein

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The complete coding dictionary reveals that of the possible 64 triplet codons, 61 in code the 20 amino acids found in proteins, while three triplets terminate translation. One of the 61 amino acid coding triplets is the initiation code on and specifies methionine.

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The complex multistep process begins with the transfer of genetic information stored in DNA two RNA

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The use of RNA homopolymers and hetero polymers in a cell free protein synthesizing system allows the determination of the composition, but not the sequence, of triplet codons designating specific amino acids.

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Transcription the initial step in gene expression is the synthesis under the direction of RNA polymerase of a strand of RNA complementary to a DNA template.

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Transcriptional activators and repressors bind to enhancer and silencer elements and regulate transcription initiation by aiding or preventing the assembly of preinitiation complexes and the release of RNA polymerase 2 from pre initiation into full transcription elongation

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Use of the triplet binding assay and of repeating copolymers allow the determination of the sequences of triplet codons designating specific amino acids.

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When discussing transcription, the DNA strand that serves as a template for RNA polymerase is denoted as the template strand in the complementary DNA strand is called the coding strand.

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Whereas bacterial M RNAs are able to be translated immediately, the initial transcripts of eukaryotes must undergo RNA processing steps that include adding a 5' m7G app, splicing out noncoding sequences called introns, and adding a 3' pol-A tail make the mature mRNA

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While in bacteria the base sequence of DNA is transcribed into M RNA that is immediately and directly translated into the amino acid sequence as dictated by the genetic code, eukaryotic M RNA's require significant alteration before they are transported to the cytoplasm and translated

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While the AAUAAA signal sequence is not found on all eukaryotic mRNA's it appears to be essential to those that have it. If the sequence is changed as a result of mutation, those transcripts that would normally have it cannot add the poly A tail in the absence of this till these RNA transcripts are rapidly degraded by nucleases

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because the interaction of promoters with RNA polymerase governs the efficiency of transcription by regulating the initiation of transcription, important self promoter sequences cannot be overemphasized.

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in short an unstable DNA enzyme complex is formed during the initiation of transcription, stability is established once elongation manages to create a DNA RNA duplex of sufficient size, elongation proceeds, and then enzyme stability again characterizes termination of transcription. As you think back on this cycle try to visualize the process mentally, from the time of the DNA first associates with the enzyme until the transcript is released from the large molecular complex.

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protein coding genes are often expressed at different times, in response to different signals, and in different cell types

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some introns are self splicing and catalyze their own removal from the primary transcript. However, most eukaryotic introns are removed by an RNA/protein complex called the spliceosome.

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the Poly a binding protein as the name suggests binds to poly A tails and prevents nucleuses from degrading the 3' end of the mRNA it is also important for the exportation of the mRNA from the nucleus to the cytoplasm and for translation of the mRNA

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the general transcription factors are essential because an RNA polymerase two combined directly to eukaryotic core promoter sites and initiate transcription without their presence the general transcription factors involved with human RNA polymerase 2 bonding are well characterized and are designated TFIIA, TFIIA, and so on.

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these observations suggested that genetic information, stored in DNA, is transferred to an RNA intermediate, which directs the synthesis of proteins.

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Although eukaryotic promoter elements can determine the site and general efficiency of initiation, other elements known as enhancers and silencers have more dramatic effects on eukaryotic gene transcription. As their names suggest, enhancers increase transcription levels and silencers decrease them. The locations of these elements can vary from immediately upstream of a promoter to downstream within or kilobases away from a gene

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In 1985 exceptions to the standard coding dictionary were also discovered in the bacterium mycoplasma capricolum, and in the nuclear genes of the protozoan ciliates Paramecium, Tetrahymena, and Stylonychia.

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at least four different types of cis-acting DNA elements regulate the initiation of transcription by RNA polymerase II.

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To prove 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, I'd independently discovered such a molecule in rat liver called RNA polymerase

Third Point about polymerase-promoter interactions involves the Sigma subunit in bacteria.

Each Sigma factor recognizes different promoter sequences, which in turn provides specificity to the initiation of transcription

once RNA polymerase has recognized and bound to the promoter DNA is locally converted from its double stranded form to an open structure, exposing the template strand

the enzyme then proceeds to initiate RNA synthesis, whereby the first ribonucleoside triphosphate which is complementary to the first template nucleotide, is inserted at the start site this will be the 5' end of the transcript as noted earlier unlike the DNA synthesis no primer is required

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

RNA

the process by which RNA molecules are synthesized on a DNA template is called

Transcription it results in an M RNA molecule complementary to the gene sequence of one of the 2 strands of the double Helix each triplet codon in the mRNA is in turn complementary to the anticodon region of its corresponding tRNA, which inserts the correct amino acid into the polypeptide chain during translation

The enzyme traverses the entire gene until eventually it encounters a specific nucleotide sequence that acts as a termination signal

an interesting aspect of termination and bacteria is that the termination sequence alluded to above is actually transcribed into RNA causing it to fold back on itself forming what is called a hairpin secondary structure which is held together by hydrogen bonds

After triplet sequence in tRNA that is complementary to a code on of mRNA is known as a

anticodon

With several exceptions, each group of three ribonucleotides, called a _________, specifies one amino acid, making the code nearly ________________.

codon, unambiguous

The code is ___________; the order of codons in the mRNA determines the order of amino acids in the encoded protein.

colinear

No internal punctuation (analogous, for example, to a comma) is used in the code. Thus, the code is said to be _______________. Once translation of mRNA begins, the codons are read one after the other with no breaks between them (until a stop signal is reached).

commaless

Hair Pin Secondary Structure

common type of secondary structure in RNA molecules. In RNA, the secondary structure is the basic shape that the sequence of A, C, U, and G nucleotides form after they are linked in series, such a folding or curling of the nucleic acid strand.

The code is _____________, meaning that a given amino acid can be specified by more than one triplet codon. This is the case for 18 of the 20 amino acids.

degenerate

RNA editing

direct alteration of one or more nucleotides in an mRNA that has already been synthesized

holoenzymes

enzymes containing their cofactors

In bacteria, the process of transcription is similar to, but less complex, than ____________, where the initial transcript must be processed prior to its translation.

eukaryotes

In some cases the trip of binding was inefficient and assignments were not possible

eventually about 50 of the 64 possible triplets were assigned

Yet another innovative technique for deciphering the genetic code was developed in the early 1960s by Har Gobind Khorana, he was able to chemically synthesize long RNA molecules consisting of short ribonucleotide sequences repeated many times

first he created the individual short sequences (di-, tri-, and tetranucleotides); And he replicated them many times and finally joined them enzymatically to form the long polynucleotides referred to as copolymers

A similar situation has been observed in other viruses and bacteria. The employment of overlapping reading frames optimizes the limited amount of genetic material present

however such an approach to storing information has a distinct disadvantage in that a single mutation may affect more than one protein and thus increase the chances that the change will be deleterious or lethal.

Substitution editing

individual nucleotide bases are altered

transcriptional activators and transcriptional repressors

influence the efficiency or the rate of RNA polymerase 2 transcription initiation

The code contains one "start" and three "stop" signals, triplets that __________ and __________ translation, respectively.

initiate and terminate

approximal promoter element

is located upstream of the start site and helps modulate the level of transcription

RNA polymerase

it has the same general substrate requirements as does DNA polymerase, the major exception being that the substrate nucleotides contain the ribose rather than the deoxyribose from the sugar unlike the DNA polymerase, no primer is required to initiate synthesis of the overall reaction

MS2 is a virus that infects the bacterium E.coli.

it's genetic it's a single stranded RNA it's genome (one of the smallest known) insists of only about 3500 rather than nucleotides making up only four genes these genes specify a coat protein, an RNA-directied replicase, a lysis protein the simple system of a small genome and few gene products allowed Fiers and his colleagues to sequence the genes and their products

In some eukaryotic genes, a cis-acting element within the core promoter is the Goldberg-Hogness Box, or TATA box

it's located about 30 nucleotides pairs upstream from the start point of transcription

The structure of DNA consist of a

linear sequence of deoxyribonucleotides this sequence ultimately dictates the composition of proteins, the end products of protein-coding genes

Francois Jacob and Jacques Monod postulated the existence of

messenger RNA (mRNA)

Experiment

multiple experimental syntheses were conducted with each homopolymer they always made all 20 amino acids available, but for each experiment they attached a radioactive label to a different amino acid and thus could tell when that amino acid had been incorporated into the resulting polypeptide. For example, from experiments using C-phenylalanine, Nirenberg and Matthaei concluded that the RNA homopolmer UUUUU... (polyurylidic acid or poly U) directed the incorporation of only phenylalanine into the peptide homopolymer polyphenylalanine. Assuming the validity of a triplet code, they made the first specific codon assignment: UUU codes for phenylalanine. Running similar experiments, they quickly found that AAA codes for lysine and CCC codes for proline.

The second point is that 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 in bacteria, both strong promoters and weak promoters have been discovered, causing a variation in time of initiation from once every one to two seconds to as little as once every 10 to 20 minutes

mutations and promoter sequences may severely reduce the initiation of gene expression

The code is _________. After translation commences, any single ribonucleotide with the mRNA is part of only one triplet.

nonoverlapping

Insertion/deletion editing

nucleotides are added/deleted from the total number of bases

unlike bacteria there is no specific sequence that signals for the termination of transcription in fact RNA polymerase two often continues transcription well beyond what will be the eventual 3' end of the mature mRNA

once transcription as incorporated a specific sequenceAAUAAA known as the polyadenylation signal sequence the transcript is enzymatically cleaved roughly 10 to 35 bases further downstream in the 3' direction the clamp will then open and both DNA and RNA are released from the enzyme as transcription is terminated this completes the cycle that constitutes transcription

Degenerate

one amino acid may be specified by more than one triplet

transcription results in the synthesis of a single stranded RNA molecule complementary to a region along

only one of the two strands of DNA double Helix

In contrast, to RNA polymerase (which constructs mRNA in vitro)

polynucleotide phosphorylase does not require a DNA template as a result, the order in which ribonucleotides are added to a growing RNA is a random, depending only on the relative concentrations of the four ribonucleoside diphosphates present in the reaction mixture.

like DNA polymerase RNA polymerase can perform proofreading as it adds each nucleotide

proofreading leads to the recognition of mismatches where a non-complementary base has been inserted in such a case the enzyme backs up and removes the mismatch it then reverses direction and continues elongation

Several codons act as signals that initiate or terminate

protein synthesis

The gain or loss of letters shifts the ____________ during translation

reading frame

small nuclear ribonucleoproteins (snRNPs)

remove introns and splice exons together

in vitro, in the presence of high concentrations of ribonucleoside diphosphates, the reaction can be "forced" in the opposite direction, to

synthesize RNA

core enzyme

the enzyme responsible for catalysis in a multipart holoenzyme

Small nuclear RNAs (snRNAs)

A class of small RNA molecules, located in the nucleus, some of which are components of the spliceosome.

Sydney Brenner argued on theoretical grounds that the code has to be triplet base since three letter words represent the minimal use of four letters to specify amino acids.

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

intrinsic termination

A mechanism in prokaryotes that involves the formation of a G-C rich hairpin loop followed by 6 or more U's in a RNA and results in the termination of transcription and release of the RNA transcript.

overlapping genes

two or more genes in which part or all of one gene is embedded in the other

The code is nearly __________. With only minor exceptions, a single coding dictionary is used by almost all viruses, bacteria, archaea, and eukaryotes.

universal

It was proposed by James Darnell and others that this primary transcript of a gene (a pre-mRNA) must be processed in the nucleus before it appears in the cytoplasm as a mature mRNA molecule.

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Initiation of gene transcription is dependent on an upstream (5') DNA region, called a promoter, which represents the initial binding site for RNA polymerase. Promoters contain specific DNA sequences, such as the -10 site (Pribnow box) and bacteria in the TATA box in eukaryotes, which are essential to polymerase binding.

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1. some genes can encode for more than one protein products through the alternative use of exons this process known as alternative splicing produces different mature mRNAs from the same pre-mRNA by splicing out introns and ligating together different combinations of exons. This means that a eukaryotic genome can encode a greater number of proteins than it has protein coding genes 2. Introns may also be important to the evolution of genes. On evolutionary timescales, the DNA sequences may be moved around within the genome. The modular exon slash intron gene structure allows for a phenomenon known as exon shuffling whereby new genes may evolve when an exon is introduced into an existing gene 3. Once an intron is excised from a pre mRNA it is generally degraded. However, there are many documented cases where an intron actually contains a non-coding RNA, such as a micro-RNA (miRNA) which is a small RNA that regulates gene expression. In such cases, the excised intron is processed to liberate the non-coding RNA, which then functions within the cell 4. Introns can also regulate transcription. For example, intronic sequences in the DNA frequently harbor cis regulatory elements, such as enhancers and silencers that upregulate and downregulate transcription respectively.

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An enzyme known as polya polymerase then catalyzes the addition of a poly- A tail to the free 3' OH group at the end of almost all mRNA's studied in a variety of eukaryotic organisms

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Confirmation for the coding dictionary, including codons for initiation and termination, was obtained by comparing the complete nucleotide sequences of phage MS2 for the amino acid sequence of the corresponding proteins. Other findings support the belief that, with other minor exceptions, the code is universal for all organisms.

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Early studies of the genetic code revealed it to be triplet in nature and to be non-overlapping, commaless, and degenerate

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Even while the genetic code was being studied, it was quite clear that proteins were the end products of many genes hence while some geneticists were attempting to elucidate the code other research efforts were directed towards the nature of genetic expression.

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Their succes was dependent on the use of 2 experimental tools

1. a cell free (in vitro) system for synthesizing proteins and 2. a means or producing synthetic mRNA's to serve as templates for polypeptide synthesis in the cell-free system

we might envision the simplest possible mechanism for removing an intron to involve 2 steps

1. the intron is caught at both ends by an endonuclease and 2. the adjacent axons are joined or ligated by a ligase

One example of specific assignments made in this way will illustrate the value of khorana's approach. Consider the following experiments in concert with one another:

1. there are repeating trinucleotide sequence UUCUUCUUC ... can be read as three possible repeating triplets—UUC, UCU, and CUU-- depending on the initiation point. when placed in a self retranslation system three different polypeptide homopolymers-- containing phenylalanine, serene, or leucine-- our 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... produces 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 in common to both experiments, must encode either leucine or serine but not phenylalanine. Thus either CUU or UUC and codes leucine or S, while other encodes phenylalanine. 3. to derive more specific information, we can examine the results of using the repeating tetranucleotide sequence UUAC, which produces the triplets UUA, UAC, ACU, and CUU. The CUU triplet is 1 of the two in which we are interested. 3 amino acids are incorporated by this experiment: leucine, threonine, and tyrosine. Because CUU Must specify only serine or leucine and because of these two only leucine appears in the resulting polypeptide we may conclude that CUU specifies leucine. once this assignment is established we can logically determine all others. of the two triplet pairs remaining(UUC and UCU from the first experiment and UCU and CUC from the second experiment), whichever triplet is common to both must encode serine. This is UCU. By elimination, UUC mined to encode phenylalanine and CUC is determined to encode leucine.

Much of our knowledge of transcription has been derived from studies of bacteria. Most of the general aspects of the mechanisms of these processes are similar in eukaryotes, but there are several notable differences

1. transcription and eukaryotes occurs within the nucleus. Thus, unlike the bacterial process, and eukaryotes the RNA transcript is not free to associate with ribosomes prior to the completion of transcription. For the mRNA to be translated, it must move out of the nucleus into the cytoplasm. 2. Transcription in eukaryotes occurs under the direction of three separate forms of RNA polymerase, rather than the single form seen in bacteria. 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 chromatin remodeling reflects the dynamics involved in the conformational change that occurs as the DNA Helix is opened 4. initiation in 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 Sigma subunit to bind the promoter and initiate transcription, and eukaryotes several general transcription factors are required to bind the promoter recruit RNA polymerase and initiate transcription. Furthermore in addition to promoters eukaryotic genes often have other cis-acting control units called enhancers and silencers which greatly influenced transcriptional activity 5. 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 involve sequence specific cleavage of the transcript, which then leads to eventual dissociation of RNA polymerase from the DNA template 6. in eukaryotes the initial or primary transcripts of protein coding mRNA's, called pre-mRNA's, undergo complex alterations, generally referred to as "processing" to produce a mature mRNA. Processing often involves the addition of a5' cap and 3' tail, and the removal of intervening sequences that are not a part of the mature mRNA.

there are ______ triplet codons assigned to _____ different amino acids

26, 10

Genetic code is written in units of

3 letters triplets of ribonucleotides in mRNA that reflect the stores information in genes most of the triplets direct the incorporation of a specific amino acid into a protein as synthesized

Splice acceptor

3' end of intron

ORF (open reading frame)

Any DNA sequence that produces a functional mRNA, one with a start and stop code on between which is a series of triplet codons specifying the amino acids making up a polypeptide

Suppose that only A and C are used for synthesizing the mRNA, in a ration of 1A : 5C. The insertion of a ribonucleotide at any position along the RNA molecule during its synthesis is determined by the ration of A:C. Therefore, there is a 1/6 possibility 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

By examining the percentages of the different amino acids incorporated into the polypeptide synthesized under the direction of the mRNA just described, we can propose probable base compositions for the codons specifying eachof those amino acids Using as many as all four ribonucleotides to construct mRNAs, the researchers conducted many similar experiments. Although the determination by this means of the composition of triplet code words corresponding to all 20 amino acids represented a very significant breakthrough, the specific sequences of triplets were still unknown- other approaches were still needed.

several 1979 reports on the coding properties of DNA derived from mitochondria (mtDNA) from yeast and humans began to undermine the hypothesis of the universality of the genetic language

Cloned mtDNA fragments were sequenced and compared with the amino acid sequences of various mitochondrial proteins revealing several exceptions to the coding dictionary

exons

Coding segments of eukaryotic DNA.

Transcription factors

Collection of proteins that mediate the binding of RNA polymerase and the initiation of transcription.

Genetic information is stored in _________ and encoded in a from that is nearly universal in all living things on Earth.

DNA

consensus sequences

DNA sequences that are similar (homologous) and different genes of the same Organism or in one or more genes of related organisms their conservation during evolution attests to the critical nature of their role in biological processes two consensus sequences have been found in bacterial promoters. One, TATAAT, is located 10 nucleotides upstream from the site of initial transcription mutations in either region diminish transcription often severely

Experimental evidence supporting the triplet nature of the code was subsequently derived form research by

Francis Crick and his colleagues.

Earlier it was stated that the genetic code is non overlapping, meaning that each ribonucleotide in the code for a given polypeptide is part of only one codon. However this characteristic of the code does not rule out the possibility that a single mRNA may have multiple initiation points for translation.

If so these points could theoretically create several different reading frames within the same mRNA thus specifying more than one polypeptide this concept creates overlapping genes.

In molecular genetics, then, cis- elements or regulatory sequences located within the same molecule of DNA as the target they regulate.

In Contrast, transacting factors are proteins that influence gene expression by binding cis-acting elements

RNA polymerase I and III 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.

In contrast, RNA polymerase III rich transcribes protein coding genes is highly regulated

Note, the specific triplet codon assignments were possible only because homopolymers were used.

In this method, only the general nucleotide composition of the template is known, not the specific order of nucleotides in each triplet but since three identical letters can only have one possible sequence three of the actual codons for phenylalanine, lysine and proline could be identified

core promoter

Includes the transcription start site it determines where RNA polymerase 2 binds to the DNA and where it begins transcribing the DNA to RNA

Gene expression

Information present on one of the two strands of DNA (the template strand) is transferred into an RNA complement through the process of transcription. Once, synthesized this RNA acts as a "messenger" molecule, which in eukaryotes out of the nucleus The mRNAs then associate with ribosomes, where decoding into proteins occurs.

Enhancers and silencers

Is the efficiency or the rate of transcription initiation by RNA polymerase 2 from the core promoter element

The use of two tetranucleotide sequences, GUAU 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 to detect there are no triplets common to both messages in both seem to contain at least one triplet that terminates protein synthesis

RNA's four ribonucleotides, analogous to an alphabet of four "letters," can be arranged into 64 different three-letter sequences....

Most of these triplet codons in RNA encode one of the 20 amino acids present in proteins.

introns

Noncoding segments of nucleic acid that lie between coding sequences.

splice donor

Occurs where 3' end of exon hits 5' end of intron... typically GU followed by purines

RNA splicing

Process of removing introns from a pre mRNA and joining together exons

gRNA (guide RNA)

RNA molecule that serves as a template for an alteration made in mRNA during RNA editing

Once secured by the clamp the two strands of a small region of duplex DNA separate at a position within the enzyme referred to as the active center and the template strand is scanned for the transcription start site

RNA synthesis is then initiated on the DNA template strand however the entire complex remains unstable and transcription usually terminates following the incorporation of only a few ribonucleotides Transcription at this point is said to have achieved a level of highly processive RNA polymerization meaning that the enzyme is able to catalyze numerous consecutive polymerization reactions without releasing its substrate as transcription proceeds the enzyme moves along the DNA and at any given time about 40 base pairs of DNA in 18 residues of the growing our name chain are part of the enzyme substrate complex

ribozymes

RNAs that are capable of catalytic activity

Rho dependent termination

Rho protein recognizes specific DNA sequences and causes a pause in the RNA polymerase

Nirenberg and Leder test

The amino acid to be tested was made radioactive and added to the cell lysate. Enzymes in the lysate attracted the radioactive amino acid to its cognate tRNA, creating a "charged" tRNA. Because code on compositions (though not exact sequences) were known it was able to narrow the decision as to which amino acids should be tested for each specific triplet. The radioactively charged tRNA, the RNA triplet, and ribosomes were incubated together and then applied to a nitrocellulose filter... The filter retains ribosomes (because of their larger size) but not the other smaller components such as charge tRNA if radioactivity was not retained on the filter and incorrect amino acid had been tested if radioactivity remained on the filter it did so because the charge tRNA add ground to the RNA triplet associated with the ribosome which itself remained on the filter in such a case a specific code on assignment could be made.

Central Dogma of Molecular Genetics

The directional flow of genetic informaiton from DNA to RNA to protein

In a cell-free protein-synthesizing system, amino acids are incorporated into polypeptide chains in a test tube

The process begins with a cell extract, or lysate, containing all the essential factors for protein synthesis: ribosomes, tRNAs, amino acids, and other molecules essential to translation but with organelles and cell membranes removed this allows scientists to follow or "trace" the progress of protein synthesis , one or more of the amino acids must be radioactive finally, mRNA must be added to serve as the template to be translated.

Nirenberg, Matthaei, and Ochoa turned to the use of mixed RNA heteropolymers in which 2, 3, or 4 different ribonucleoside diphosphates were used in combination to form the artificial message

The researchers reasoned that if they knew the relative proportion of each type of ribonucleoside diphosphate in the synthetic mRNA, they could predict the frequency of each of the possible triplet codons it contained. If they added they then added the mRNA to the cell-free system and ascertained the percentage of each amino acid present in the resulting polypeptide, they could then analyze the results and predict the composition of the triples that had specified those amino particular amino acids.

As with most new ideas in molecular genetics, the initial supporting experimental evidence for an RNA intermediate was based on studies of bacteria and bacteriophages.

The results of these experiments agreed with the concept of messenger RNA (mRNA) being made on a DNA template and then directing the synthesis of specific proteins and association with ribosomes

Genetic code is written in linear form, using as "letters" the bases in ribonucleotides that compose mRNA molecules.

The ribonucleotide sequence is derived from the complementary nucleotide bases in DNA.

DNA sequences such as these located near the region to be transcribed are said to be CIS acting elements

The term cis drawn from organic chemistry nomenclature, means "next to" or on the same side as other functional groups, and contrast to being trans two or "across from"

the code is triplet in nature degenerate, unambiguous, and commaless although it contains punctuation in the form of start and stop symbols.

These individual principles have been confirmed by the detailed analysis of the RNA containing bacteriophage MS2 by Walter Fiers and his coworkers

The probability of the insertion of a specific ribonucleotide is proportional to the availability of that molecule relative to other available ribonucleotides

This point is absolutely critical to understanding the work or Nirenberg and others in the ensuing discussion together the cell-free system for protein synthesis and the availability of synthetic mRNA's provided a means of deciphering the ribonucleotide composition of various codons encoding specific amino acids

Poly g was not a functional template, probably because the molecules folds back on itself, likely blocking association with the ribosome.

Thus, the assignment for GGG had to await other approaches.

Each "word" within an mRNA consists of three ribonucleotide letters, thus referred to as a

Triplet code

Splicesome

a complex of specialized RNA and protein subunits that removes introns from a transcribed pre-mRNA segment.

Transcription is dependent on

a major polymerase enzyme and a cast of supporting proteins

Unambiguous

a single code on specifies only one amino acid

a dinucleotide made in this way is converted to an mRNA with two repeating triplet codons

a trinucleotide is converted into an mRNA that can be read as containing one of the three potential repeating triplets depending on the point at which initiation occurs a tetranucleotide creates a message with four repeating triplet sequences

general transcription factors

absolutely required for all RNA polymerase to mediated transcription

the identification of introns and eukaryotic genes involves a direct comparison of nucleotide sequences of DNA with those of M RNA and their correlation with amino acid sequences

action approach allows the precise identification of all intervening sequences by identifying common sequences that appear at intron/exon boundaries, scientists are now able to identify introns with excellent accuracy using only the genomic DNA sequence in conjunctural tools

subsequent ribonucleotide complements are inserted and linked together by phosphodiester bonds as RNA polymerization proceeds

after these initial ribonucleotides have been added to the growing RNA chain the Sigma subunit dissociates from the holoenzyme and chain elongation proceeds under the direction of the core enzyme

polynucleotide phosphorylase

allowed the artificial synthesis of RNA templates, which could be added to the cell-free system this enzyme acts metabolically in bacterial cells to degrade RNA was discovered in 1955 by Marianne Grunberg Manago, and Severo Ochoa

the significance of transcription is that it is the final 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 findings 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 did not appear to participate directly in protein synthesis. 2. RNA is synthesized in the nucleus of eukaryotic cells, in which DNA is found and is chemically similar to DNA 3. following its synthesis most messenger RNA migrates to the cytoplasm, in which protein synthesis (translation) occurs

the various techniques applied to decipher the genetic code have yielded a Dictionary of 61 triplet codons assigned to amino acids.

the remaining 3 codons are termination signals not specifying any amino acid.

the initial step in bacterial gene transcription is referred to as template binding

the sight of this initial binding is established when the RNA polymerase Sigma subunit recognizes specific DNA sequences called promoters these recognition sequences are located in the 5' region, upstream from the coding sequence of a gene it is believed that a holoenzyme "explores" a length of DNA until it encounters a promoter region and binds there 2 about 60 nucleotide pairs along the double Helix, 40 of which are upstream from the point of initial transcription once this occurs, the Helix listing nature, or unwound, locally, making the the template strand over DNA accessible to the enzyme the point at which transcription begins is called the transcription start site, often indicated as position +1.

Nirenberg and Mattahaei synthesized RNA homopolymers

these are RNA molecules containging only one type of ribonucleotide in other words, the mRNA they added to their cell-free protein-synthesis-sizing system was either UUUUUU..... AAAAAA..... CCCCCC.... or GGGGGG They tested each of these to see which, if any, amino acids were consequently incorporated into newly synthesized proteins.

Marshall Nirenberg and J Heinrich Matthaei became the first to link specific coding sequences to specific amino acids

this laid a cornerstone for the complete analysis of the genetic code

Neirenberg and Philip Leder developed The triplet binding assay, leading to specific sequence assignments for triplet codon

this technique took advantage of the observation that ribosomes when presented in vitro with an RNA sequence as short as three ribonucleotides, will bind to it and form a complex similar to what is found in vivo

The genetic code is initially transferred from DNA to RNA, in the process of ____________. Once transferred to RNA, the genetic code exists as triplet codons, which are sets of three ribonucleotides in which each ribonucleotide is one of the four kinds composing RNA.

transcription

the triplet RNA sequence acts like a code on an mRNA attracting a ____________ molecule containing a complementary sequence and carrying a specific amino acid

transfer RNA (tRNA)

the amino acid sequence of the MS2 coat protein was completed in 1970 and the nucleotide sequence of the gene encoding the protein was reported in 1972.

when the chemical compositions of this gene and its encoded protein are compared they are found to exhibit collinearity with one another that is, the linear sequence of triplet codons formed by the ribonucleotides corresponds precisely with the linear sequence of amino acids in the protein furthermore, the code on for the first amino acid is AUG, the common initiator codon; the code on for the last amino acid is followed by the consecutive termination codons, UAA and UAG.

when these synthetic mrnas were added to a cell free system the predicted proportions of amino acids were found to be incorporated in the resulting polypeptides

when these data were combined with data drawn from mixed copolymer and triplet binding experiments specific assignments were possible

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 is the code was anything other than a triplet.

Using phage T4 they studied frameshift mutations

which result from the addition or deletion of one or more nucleotides within a gene and subsequently the mRNA transcribed from it.

these discoveries led to some major conclusions

you know the code is degenerate and unambiguous