Chapter 13: The Genetic Code and Transcription Flashcards
True or False?
The genetic code is degenerate, meaning that a codon can specify more than one amino acid.
FALSE
The degeneracy of the genetic code means that an amino acid may be coded for by more than one codon. However, a single codon can only ever specify one amino acid.
A DNA sequence produces a mutant protein in which several amino acids in the middle of the protein differ from the normal protein. What kind of mutation could have occurred?
An addition and a deletion mutation
A single addition or deletion would change the reading frame of the protein, but if another mutation occurred to cancel the effects of the first mutation, only those amino acids between the mutations would change.
True or False?
A polycistronic mRNA may be transcribed if the gene products are used in the same pathway or needed at the same time.
FALSE
Polycistronic mRNAs are produced only in prokaryotes. In eukaryotes, a single gene is transcribed at a time.
The following statements about eukaryotic transcription are true…
Transcription initiation occurs when RNA polymerase binds to a complex of transcription factors at the TATA box.
Eukaryotic promoter regions contain a TATA box and a CAAT box.
The transcripts produced contain both exons and introns.
Both the codons UUU and UUC specify the amino acid phenylalanine. What is the term for this phenomenon?
Degenerate
Degeneracy of the code means that a given amino acid can be specified by more than one triplet codon.
True or False?
The code is nonoverlapping, meaning that, assuming “standard translation,” a given base participates in the specification of one and only one amino acid.
True
The genetic code is said to be triplet, meaning that there ________.
are three bases in mRNA that code for an amino acid
Which type of mutation helped lead to the understanding that the genetic code is based on triplets?
Frameshift
Insertions or deletions of one or two nucleotides resulted in frameshift mutations. Insertion or deletion of three nucleotides resulted in insertion or deletion of a single amino acid and did not shift the reading frame.
A class of mutations that results in multiple contiguous (side-by-side) amino acid changes in proteins is probably caused by which of the following type of mutation?
frameshift
Significant in the deciphering of the genetic code was the discovery of the enzyme polynucleotide phosphorylase. What is this enzyme used for?
manufacture of synthetic RNA for cell-free systems
In 1964, Nirenberg and Leder used the triplet binding assay to determine specific codon assignments. A complex of which of the following components was trapped in the nitrocellulose filter?
charged tRNA, RNA triplet, and ribosome
All of the following experiments or discoveries helped to identify the “triplet nature” of the DNA code:
Anticodons
Repeating copolymers
Frameshift mutations
True or False?
The Universality of the genetic code discovery helped to identify the “triplet nature” of the DNA code.
FALSE
The universality of the code refers to the fact that, for the most part, all the DNA code of life’s genomes can be read in the same way. In other words, a codon that represents leucine in bacteria, will also represent leucine in humans.
Introns are known to contain termination codons (UAA, UGA, or UAG), yet these codons do not interrupt the coding of a particular protein. Why?
Introns are removed from mRNA before translation.
When examining the genetic code, it is apparent that ________.
there can be more than one codon for a particular amino acid
The genetic code is fairly consistent among all organisms. The term often used to describe such consistency in the code is ________.
universal
RNA synthesis from a DNA template is called _______.
transcription
Transcription is initiated when the cell signals for the expression of a particular gene and involves the synthesis of RNA from a DNA template.
The relationship between a gene and a messenger RNA is that ________.
mRNAs are made from genes
Which subunit of RNA polymerase establishes template binding to a promoter in prokaryotes?
Sigma
The sigma subunit recognizes the promoter sequence. Different sigma subunits can be employed to regulate the expression of genes at the transcriptional level.
When considering the initiation of transcription, one often finds consensus sequences located in the region of the DNA where RNA polymerase(s) binds. Which of the following is a common consensus sequence?
TATA
True or False?
Messenger RNA is usually polycistronic in eukaryotes.
False
What are two main types of posttranscriptional modifications that take place in the mRNA of eukaryotes?
The addition of a 7-mG cap at the 5’ end of the transcript and the addition of a poly-A sequence at the 3’ end of the message.
These are the two steps in the processing of eukaryotic mRNA.
True or False?
A 5’-cap describes the addition of a base, usually thymine, to the 5’ end of a completed peptide.
False
True or False?
A 3’ poly-A tail and a 5’-cap are common components of prokaryotic RNAs.
False
True or False?
Transcription factors function to help move ribosomes along the mRNA.
False
transcription
Transfer of genetic information from DNA by the synthesis of a complementary RNA molecule using a DNA template.
messenger RNA (mRNA)
An RNA molecule transcribed from DNA and translated into the amino acid sequence of a polypeptide.
transfer RNA (tRNA)
A small ribonucleic acid molecule with an essential role in translation. tRNAs contain: (1) a three-base segment (anticodon) that recognizes a codon in mRNA; (2) a binding site for the specific amino acid corresponding to the anticodon; and (3) recognition sites for interaction with ribosomes and with the enzyme that links the tRNA to its specific amino acid.
polycistronic mRNA
A messenger RNA molecule that encodes the amino acid sequence of two or more polypeptide chains in adjacent structural genes.
frameshift mutation
A mutational event leading to the insertion of one or more base pairs in a gene,shifting the codon reading frame in all codons that follow the mutational site.
codon
A triplet of nucleotides that specifies a particular amino acid or a start or stop signal in the genetic code. Sixty-one codons specify the amino acids used in proteins, and three codons, called stop codons, signal termination of growth of the polypeptide chain. One codon acts as a start codon in addition to specifying an amino acid.
translation
The derivation of the amino acid sequence of a polypeptide from the base sequence of an mRNA molecule in association with a ribosome and tRNAs.
overlapping code
A hypothetical genetic code in which any given triplet is shared by more than one adjacent codon.
universal code
A genetic code used by all life forms. Some exceptions are found in mitochondria, ciliates, and mycoplasmas.
It has been determined that the gene for Duchenne muscular dystrophy (DMD) is more than 2000 kb (kilobases) in length; however, the mRNA produced by this gene is only about 14 kb long. What is a likely cause of this discrepancy?
The introns have been spliced out during mRNA processing.
True or False?
Heterogeneous nuclear RNA is a primary transcript in eukaryotes that is processed prior to involvement in translation.
true
Lists the steps of mRNA production in eukaryotes in the correct order:
Transcription, 5’ cap addition, addition of poly-A tail, exon splicing, passage through nuclear membrane
Which of the following is characteristic of transcription in eukaryotes but NOT in prokaryotes?
Exon splicing
Introns must be removed from eukaryotic pre-mRNA; prokaryotic mRNA does not contain introns.
Which of the following is characteristic of transcription in eukaryotes AND in prokaryotes?
A single transcript may be transcribed and translated simultaneously.
A 3’ untranslated trailer sequence
A 5’ untranslated leader sequence
Which of the following best describes the function of the 5’mRNA cap?
It provides a site for ribosome binding in the cytoplasm.
The 5’ cap is essential for recognition of the mRNA by ribosomes in the cytoplasm.
What is a characteristic of RNA splicing in Eukaryotes?
Exon/intron boundaries are typically characterized by a 5’ GU splice junction and a 3’ AG splice junction.
These splice junctions are recognized by the spliceosome so that accurate removal of introns is possible.
A snRNP is best described as _______.
small RNAs associated with protein complexes in the nucleus
snRNPs recognize the 5’ and 3’ splice junctions and the branch point sequence, excise the intron, and splice together the exons.
Which of the following is most likely attributable to a base substitution at a 5’ splice junction?
A longer than usual final transcript
Such a mutation could block intron removal, resulting in a longer than usual transcript.
Which of the following contains the three posttranscriptional modifications often seen in the maturation of mRNA in eukaryotes?
5’-capping, 3’-poly(A) tail addition, splicing
Of the following three types of nucleic acids–DNA, mRNA, tRNA–which is most likely to contain modified bases?
tRNA
Central Dogma
DNA»_space; RNA»_space; Protein
Genetic Code characteristics
Code is in linear form
RNA sequence is derived from complementary bases of DNA
Each “word” of the code in mRNA contains 3 ribonucleotide “letters”
Each group of 3 ribonucleotides is called a CODON; a codon specifies one amino acid
Therefore, the code is TRIPLET
Genetic Code characteristics cont…..
The code is unambiguous, each triplet specifies a single amino acid
The code is degenerate, more than one triplet can code for the same amino acid
The code contains “start” and “stop” signals
Translation of mRNA is continuous
The code is nonoverlapping
The code is “nearly” universal
How was the code determined?
How do four nucleotides encode the 20 amino acids?
Evidence supported a nonoverlapping code
1961: Jacob and Monod postulated the mRNA
mRNA was discovered, the code that is translated is in mRNA
What was the code?
Theoretical argument of a triplet
3 letters represents the minimum to encode information to specify 20 amino acids
4^3 =64 different possible combinations
4^2= 16 not enough, 4^4=256 too much
First evidence of triplet nature of code
Francis Crick were involved in this
Insertion & Deletion mutations in T4 bacteriophage
Used intercalating agents, get into the stacked bases of DNA causing insertions or deletions upon replication
This insertion or deletion causes the reading frame to shift: frameshift mutation
Treat the mutants again with intercalating agents could result in reversal of the mutant phenotype
One + and one - : normal phenotype
+++ and — : normal phenotype: supports triplet nature of the code
What if Overlapping triplet code?
Consider a sequence: GTACA
If the triplet code were overlapping, then GTA, TAC, and ACA are possible reading frame codons of this sequence
If overlapping, it restricted the amino acids that would be adjacent to the amino acid encoded by the central triplet
If true, then sequence of tripeptides would be limited, but that was not the case when peptide sequences were examined
Other arguments against an overlapping code
Point mutations would affect more than one amino acid, but this was not observed
Crick argued for an adaptor molecule, not consistent with an overlapping code
BOTTOM LINE: Evidence did NOT support an overlapping code
CONCLUSION: The code is NONOVERLAPPING
Deciphering the “Code”
1961: Nirenberg and Matthaei characterized the first specific coding sequences:
In vitro system (cell-free) that could synthesize protein
Enzyme (polynucleotide phosphorylase) that produced synthetic mRNAs, which serve as template for polypeptide synthesis in cell free system
Polynucleotide phosphorylase can be made to synthesize RNA in vitro
The formation of this RNA is random, based on the concentration of the four ribonucleoside diphosphates added to the in vitro system
Therefore; the probability of the insertion of a particular ribonucleotide is proportional to its availability in the system
THIS PROVIDES A MEANS TO DECIPHER THE CODE
Simplest Experiments
Homopolymers
Synthesize RNA homopolymers consisting of only one of the ribonucleotides
Eg., UUUUUUU…, AAAAAAA….., CCCCCC….., GGGGGGGGG…….
Radioactively label each of the 20 amino acids
Determine what amino acids these homopolymers specify
polynucleotide
A linear sequence of 20 or more nucleotides, joined by 5’-3’ phosphodiester bonds.
Homopolymer results
UUU: Phenylalanine
AAA: Lysine
CCC: Proline
GGG: no result in the early experiments
Heteropolymers
Next moved to heteropolymers with known concentrations (proportions) of each ribonucleotide
Could predict the frequency of triplets based on proportions
Match the proportion of amino acids incorporated with the proportion of potential triplets
Heteropolymer example
Produce RNA with 1A:5C ratio
Insertion of A or C at any particular position is determined by ratio of A:C
Eg., frequency of AAA = 1/6 * 1/6 * 1/6 = (1/6)^3 = 0.4%
Eg., frequency of AAC, ACA, CAA = (1/6)^2 * (5/6)= 2.3% each
Next: examine the percentages of incorporation of any given amino acid
Propose what triplet might encode that amino acid
Eg., proline seen 69% of the time: = CCC (57.9%) + 2C1A (11.6%), determines the composition of the triplet coding for an amino acid, not the specific sequence of the triplet yet
Conduct many experiments of this type to work out the code!
Triplet Binding Assays
1964: Nirenberg and Leder
Led to specific assignments of triplets
Single triplets could be bound by ribosomes
Leads to binding of anticodon
Experimental technique eventually led to determination of specific triplets coding for 50 of 64 codons
Established degeneracy of the code: >1 codon for 18 of 20 amino acids
An example of the triplet-binding assay. The UUU triplet acts as a codon, attracting the complementary tRNAPhe anticodon AAA.
Radioactively labeled amino acid, linked to tRNA.
Previous work had narrowed down what amino acids should be tested for each specific codon.
If codon specified particular anticodon it would be bound up in ribosome and this large complex would be bound to filter, smaller components would not be bound and would be washed away, tells you specific codon assignment
Repeating copolymers
Di, tri, and tetra repeats
Confirmed codons already were established, filled in the remaining gaps
Also established termination signals, code for NO amino acid incorporation
Final codon table
61 triplet codons that specify amino acids
3 termination codons, stop amino acid incorporation
deletion
A chromosomal mutation, also referred to as a deficiency, involving the loss of chromosomal material.
Degeneracy of Code
Code is degenerate: almost all amino acids are encoded by 2, 3, or 4 codons
Three amino acids are (serine, arginine, leucine) encoded by 6 codons
Only 2 (tryptophan and methionine) encoded by a single codon
There are 3 termination codes
Wobble hypothesis
Pattern of degeneracy
Third letter of the code is usually the one that is different:
3rd position free to “wobble”
First 2 positions are most critical
Allows 1 anticodon of tRNA to pair with >1 codon in mRNA
anticodon
In a tRNA molecule, the nucleotide triplet that binds to its complementary codon triplet in an mRNA molecule.
wobble hypothesis
An idea proposed by Francis Crick, stating that the third base in an anticodon can align in several ways to allow it to recognize more than one base in the codons of mRNA.
The genetic code is “nearly” universal
In some organisms or in mitochondrial genomes, the codon usage is different
Eg. “normal” UGA is a termination codon, in human & yeast Mitochondrial genomes codes for insertion of tryptophan
Other exceptions: Mycoplasma, Paramecium, Tetrahymena, & a few other organisms
Overlapping Genes
Codons are still NOT overlapping
Start initiation of transcription at different locations
Result is different reading frames produce more than one polypeptide
Seen in viruses, eg. X174
Transcription
Synthesizes RNA from a DNA template
First step in the transfer of information from DNA (nucleic acid) to protein (amino acids)
Transcription of DNA results in an mRNA molecule complementary to the gene sequence from one of the 2 DNA strands
The evidence for an RNA intermediate in Transcription
DNA is in the nucleus
But, protein synthesis occurs outside the nucleus
RNA made in the nucleus, where DNA is located
Most RNA moves to cytoplasm following synthesis
Amount of RNA is proportional to protein in cells
Experimental evidence of mRNA
1956-58: again, use a model system: bacteriophage
Use 32P to label newly made RNA following bacteriophage infection of E. coli
Base composition of newly-made RNA resembled phage, not the bacteria
Production of RNA preceded production of protein
More phage/bacteria expts: 1961
Led to Jacob and Monod’s model of gene regulation in bacteria
Model included mRNA as an intermediate made on a DNA template
RNA polymerase
If mRNA is made from DNA, then there must be an enzyme to do the job
Weiss et al.: discovered an enzyme in 1959 from rat liver that could synthesize RNA from a DNA template
RNA polymerase holoenzyme
5 subunits
2 subunits (ß & ß’) catalyze transcription
Another subunit (σ) involved in initiation of transcription
Think about the overall similarity to DNA polymerase
Transcription Initiation
RNA polymerase recognizes specific sequences in the DNA molecule called promoters
Promoters can be strong or weak
TATAAT box (Prinbrow box) 10 bases upstream of initial transcription location
Another promoter farther upstream (-35 region)
Promoters are conserved regions
chain elongation, after the sigma subunit has dissociated from the transcription complex,
then the enzyme moves along the DNA template.
Eukaryotic transcription
Transcription occurs in the nucleus
There are 3 major forms of RNA polymerases
More complicated upstream regulation of transcription
Promoters and enhancers involved
mRNAs of eukaryotes are further processed
Posttranscriptional RNA processing in eukaryotes: Heterogeneous nuclear RNA (hnRNA) is converted to messenger (mRNA), which contains a 59 cap and a 39-poly-A tail, which then has introns spliced out.
RNA polymerase
An enzyme that catalyzes the formation of an RNA polynucleotide strand using the base sequence of a DNA molecule as a template.
promoter element
An upstream regulatory region of a gene to which RNA polymerase binds prior to the initiation of transcription.
enhancer
A DNA sequence that enhances transcription and the expression of structural genes.
Enhancers can act over a distance of thousands of base pairs and can be located upstream, downstream, or internal to the gene they affect, a fact that differentiates them from promoters.
Eukaryotic promoters
cis (next to) and trans (across) acting elements
TATA box (cis factor)
CAAT box (cis factor)
Enhancers (cis factors)
Trans Factors (facilitate binding), eg. TFIID, TFIIA, TFIIB
CAAT box
A highly conserved DNA sequence found in the untranslated promoter region of eukaryotic genes. This sequence is recognized by transcription factors.
What is the start codon?
AUG
What are the stop codons?
UAG, UGA, UAA
DNA is transcribed to messenger RNA (mRNA), and the mRNA is translated to proteins on the ribosomes. A sequence of 3 nucleotides on an mRNA molecule is called a codon. Most codons specify a particular amino acid to be added to the growing protein chain. In addition, one codon codes for the amino acid methionine and functions as a “start” signal. Three codons do not code for amino acids, but instead function as “stop” signals.
Nearly every mRNA gene that codes for a protein begins with the start codon, AUG, and thus begins with a methionine.
Nearly every protein-coding sequence ends with one of the three stop codons (UAA, UAG, and UGA), which do not code for amino acids but signal the end of translation.
An amino acid sequence is determined by strings of three-letter codons on the mRNA, each of which codes for a specific amino acid or a stop signal.
The mRNA is translated in a 5’ → 3’ direction.
Follow these steps to convert a DNA sequence into an amino acid sequence.
- First, transcribe the DNA sequence to determine the mRNA sequence. Be sure to remember the following: The mRNA strand is complementary to the DNA strand. Uracil (U) takes the place of thymine (T) in RNA to pair with A on the DNA. The RNA is assembled in an antiparallel direction to the template strand of DNA. A 3’→ 5’ direction in DNA is transcribed in a 5’ → 3’ direction in RNA.
- Next, subdivide the mRNA sequence into the individual 3-letter codons in the 5’ to 3’ direction.
Before mRNA can be translated into an amino acid sequence, the mRNA must first be synthesized from DNA through transcription.
Base pairing in mRNA synthesis follows slightly different rules than in DNA synthesis: uracil (U) replaces thymine (T) in pairing with adenine (A). The codons specified by the mRNA are then translated into a string of amino acids.
Suppose that a portion of double-stranded DNA in the middle of a large gene is being transcribed by an RNA polymerase. As the polymerase moves through the following sequence of 6 bases, what is the corresponding sequence of bases in the RNA that is produced?
Coding strand from 3’ to 5’ reads C C G A G T.
Template strand from 5’ to 3’ reads G G C T C A.
Enter the sequence of bases. Begin with the first base added to the growing RNA strand, and end with the last base added.
UGAGCC
There are 3 principles to keep in mind when predicting the sequence of the mRNA produced by transcription of a particular DNA sequence.
The RNA polymerase reads the sequence of DNA bases from only 1 of the 2 strands of DNA: the template strand.
The RNA polymerase reads the code from the template strand in the 3’ to 5’ direction and produces the mRNA strand in the 5’ to 3’ direction.
In RNA, the base uracil (U) replaces the DNA base thymine (T). Thus, the base-pairing rules in transcription are A→U, T→A, C→G, and G→C, where the first base is the coding base in the template strand of the DNA and the second base is the base that is added to the growing mRNA strand.
During transcription in eukaryotes, a type of RNA polymerase called RNA polymerase II moves along the template strand of the DNA in the 3’→5’ direction. However, for any given gene, either strand of the double-stranded DNA may function as the template strand.
Which of the following initially determines which DNA strand is the template strand, and therefore in which direction RNA polymerase II moves along the DNA?
the specific sequence of bases along the DNA strands
In eukaryotes, binding of RNA polymerase II to DNA involves several other proteins called transcription factors. Many of these transcription factors bind to the DNA in the promoter region, located at the 3’ end of the sequence on the template strand. Although some transcription factors bind to both strands of the DNA, others bind specifically to only one of the strands.
Transcription factors do not bind randomly to the DNA. Information about where each transcription factor binds originates in the base sequence to which each transcription factor binds. The positioning of the transcription factors in the promoter region determines how the RNA polymerase II binds to the DNA and in which direction transcription will occur.
After transcription begins, several steps must be completed before the fully processed mRNA is ready to be used as a template for protein synthesis on the ribosomes.
These statements describe the processing that takes place before a mature mRNA exits the nucleus:
A poly-A tail (50-250 adenine nucleotides) is added to the 3’ end of the pre-mRNA.
A cap consisting of a modified guanine nucleotide is added to the 5’ end of the pre-mRNA.
Noncoding sequences called introns are spliced out by molecular complexes called spliceosomes.
spliceosome
The nuclear macromolecule complex within which splicing reactions occur to remove introns from pre-mRNAs.
Once RNA polymerase II is bound to the promoter region of a gene, transcription of the template strand begins. As transcription proceeds, three key steps occur on the RNA transcript:
Early in transcription, when the growing transcript is about 20 to 40 nucleotides long, a modified guanine nucleotide is added to the 5’ end of the transcript, creating a 5’ cap.
Introns are spliced out of the RNA transcript by spliceosomes, and the exons are joined together, producing a continuous coding region.
A poly-A tail (between 50 and 250 adenine nucleotides) is added to the 3’ end of the RNA transcript.
Only after all these steps have taken place is the mRNA complete and capable of exiting the nucleus. Once in the cytoplasm, the mRNA can participate in translation.
Promoter recognition
RNA polymerase is a holoenzyme composed of a five-subunit core enzyme and a sigma (σ) subunit. Different types of σ subunits aid in the recognition of different forms of bacterial promoters. The bacterial promoter is located immediately upstream of the starting point of transcription (identified as the +1 nucleotide of the gene). The promoter includes two short sequences, the –10 and –35 consensus sequences, which are recognized by the σ subunit.
Chain initiation:
The RNA polymerase holoenzyme first binds loosely to the promoter sequence and then binds tightly to it to form the closed promoter complex. An open promoter complex is formed once approximately 18 bp of DNA around the –10 consensus sequence are unwound. The holoenzyme then initiates RNA synthesis at the +1 nucleotide of the template strand.
Chain elongation
The RNA-coding region is the portion of the gene that is transcribed into RNA. RNA polymerase synthesizes RNA in the 5′ → 3′ direction as it moves along the template strand of DNA. The nucleotide sequence of the RNA transcript is complementary to that of the template strand and the same as that of the coding (nontemplate) strand, except that the transcript contains U instead of T.
Chain termination:
Most bacterial genes have a pair of inverted repeats and a polyadenine sequence located downstream of the RNA-coding region. Transcription of the inverted repeats produces an RNA transcript that folds into a stem-loop structure. Transcription of the polyadenine sequence produces a poly-U sequence in the RNA transcript, which facilitates release of the transcript from the DNA.
Bacterial transcription is a four-stage process.
- Promoter recognition: RNA polymerase is a holoenzyme composed of a five-subunit core enzyme and a sigma (σ) subunit. Different types of σ subunits aid in the recognition of different forms of bacterial promoters. The bacterial promoter is located immediately upstream of the starting point of transcription (identified as the +1 nucleotide of the gene). The promoter includes two short sequences, the –10 and –35 consensus sequences, which are recognized by the σ subunit.
- Chain initiation: The RNA polymerase holoenzyme first binds loosely to the promoter sequence and then binds tightly to it to form the closed promoter complex. An open promoter complex is formed once approximately 18 bp of DNA around the –10 consensus sequence are unwound. The holoenzyme then initiates RNA synthesis at the +1 nucleotide of the template strand.
- Chain elongation: The RNA-coding region is the portion of the gene that is transcribed into RNA. RNA polymerase synthesizes RNA in the 5′ → 3′ direction as it moves along the template strand of DNA. The nucleotide sequence of the RNA transcript is complementary to that of the template strand and the same as that of the coding (nontemplate) strand, except that the transcript contains U instead of T.
- Chain termination: Most bacterial genes have a pair of inverted repeats and a polyadenine sequence located downstream of the RNA-coding region. Transcription of the inverted repeats produces an RNA transcript that folds into a stem-loop structure. Transcription of the polyadenine sequence produces a poly-U sequence in the RNA transcript, which facilitates release of the transcript from the DNA.
If a mutation alters a splicing signal sequence of an intron, that intron will not be removed accurately during the splicing reaction. This will result in the production of an abnormally spliced mature mRNA.
Mutations in promoter sequences will affect transcription initiation and are likely to result in no mRNA being produced.
Show the order of events as they are thought to occur during eukaryotic transcription involving RNA polymerase II (RNA pol II).
- Transcription by RNA pol II in eukaryotes begins when TFIID recognizes and binds to the TATA box.
- The bound TFIID helps recruit TFIIB, TFIIF, and RNA pol II.
- Once those subunits of the minimal initiation complex are bound, TFIIE and TFIIH bind to form the complete initiation complex.
- Assembly of the complete initiation complex releases RNA pol II, which begins synthesizing the RNA transcript in the 5′ → 3′ direction.
- After the first 20–30 nucleotides have been synthesized, a cap consisting of a methylated guanine is added to the 5′ end of the pre-mRNA.
- Intron removal occurs as RNA pol II continues to elongate the pre-mRNA.
- When the polyadenylation signal has been transcribed, a poly-A tail is added to the 3′ end of the pre-mRNA. Polyadenylation is usually coupled with the termination of transcription.
RNA
3 major classes of RNA: Ribosomal RNA (rRNA) Transfer RNA (tRNA) Messenger RNA (mRNA)
Other RNA Classes:
Small nuclear RNAs, small nucleolar RNAs
Small interfering RNAs (siRNAs), micro RNAs (miRNAs), and others that are being discovered
Unusual nitrogenous bases found in transfer RNA.
??
Post-transcriptional modification
“Charging” tRNA
tRNA synthetases add amino acid to tRNAs
First, amino acid is converted to activated form, provides energy for transfer to tRNA
Messenger RNA (mRNA)
Transcribed by RNA polymerase II
Introns
Eukaryotic genes are often interspersed with intervening sequences
Heteroduplex
hybridization between mRNA & DNA,
areas that don’t hybridize
—>INTRONS
heteroduplex
A double-stranded nucleic acid molecule in which each polynucleotide chain has a different origin. It may be produced as an intermediate in a recombinational event or by the in vitro reannealing of single-stranded,complementary molecules.
Introns and Intron Removal
rRNA introns; Group I introns; self excised (requires cofactor)
Mitochondrial mRNA & tRNA Group II introns: also self excised (no cofactor)
Nuclear mRNA introns: removed by cell spliceosome machinery
Intron Self Excision
Found originally in Tetrahymena rRNA by Tom Cech in 1982 (Nobel prize)
Nothing but the rRNA and a cofactor is required to remove the intron, therefore the RNA is acting as an enzyme!
Steps to Intron Self Excision
First, interaction between free guanosine (cofactor) and the primary transcript
Next, the OH group of guanosine is transferred to the nucleotide next to the 5’ end of the intron
Second reaction, OH group on left exon interacts with phosphate group on 3’ end of intron
Intron spliced out, ends of exons ligated to join them
—-> mature mRNA
Ribozymes
Ribozymes: RNA molecules that act as enzymes
Hypothesis: Early life on earth was RNA based
Self-Excising Introns
Ribozymes: RNA molecules that act as enzymes
Group I Introns: found in rRNA genes, guanosine as a co-factor
Splicing mechanism involved with group I introns removed from the primary transcript leading to rRNA. The process is one of self-excision involving two transesterification reactions.
Group II Introns: self splicing, found in mRNA and tRNA of mitochondria and chloroplasts, NO co-factor required
Conserved sequences
R=A or G
Y=C or U
Intron Removal: Spliceosome
Cellular machinery to remove introns in mRNAs
Includes numerous small nuclear RNAs (snRNAs) complexed with proteins to form small nuclear riobnucleoproteins (snRNPs, snurps)
snRNAs are only found in the nucleus, Uridine rich, got the names U1, U2, etc
This processing represents a regulatory step where different introns may be alternatively removed to created alternative mRNAs
small nuclear RNA (snRNA)
Abundant species of small RNA molecules ranging in size from 90 to 400 nucleotides that in association with proteins form RNP particles known as snRNPs or snurps. Located in the nucleoplasm, snRNAs have been implicated in the processing of pre-mRNA and may have a range of cleavage and ligation functions.
Splicing mechanism involved with the removal of an intron from a pre-mRNA.
Excision is dependent on various snRNAs (U1, U2, U6) that combine with proteins to form snurps, which function as part of a large structure referred to as the spliceosome. The lariat structure in the intermediate stage is characteristic of this mechanism.
snRNAs combined with proteins: snurps
Spliceosomal removal of introns
End result: ligated exons, removed intron
Small RNA molecules in eukaryotes
Present extensively
Participate in a variety of pathways to regulate gene function and expression
Discovery led to Nobel Prize in 2006 for Andrew Fire and Craig Mello
Object of intense recent study
Source of novel treatment and investigative methodologies
Two important types of small RNAs involved in gene regulation
Small Interfering RNAs (siRNA) Micro RNAs (miRNA)
microRNA
Single-stranded RNA molecules approximately 20–23 nucleotides in length that regulate gene expression by participating in the degradation of mRNA.
Gene silencing by RNA interference (RNAi)
A protein called Dicer cleaves double-stranded RNA molecules into short interfering RNAs (siRNAs) that bind to the RNA-Induced Silencing Complex (RISC complex) for unwinding.
The single-stranded RNAs target mRNAs with complementary sequences to mark them for degradation
RNA interference (RNAi)
Inhibition of gene expression in which a protein complex (RNA-induced silencing complex,or RISC),containing a partially complementary RNA strand binds to an mRNA,leading to degradation or reduced translation of the mRNA.
The action of Dicer and RISC (RNA-induced silencing complex). Dicer binds to double-stranded RNA molecules and cleaves them into nucleotide molecules called small interfering RNAs (siRNAs). These bind to a multiprotein RISC complex and are unwound to form single-stranded molecules that target mRNAs with complementary sequences, marking them for degradation.
Binding of the catalytic domains of Dicer monomers to RNA. Domains marked with an asterisk are inactive. Cutting by the active domains produces fragments nucleotides long.
Another Role for Dicer
miRNA gene regulation
dicer
An enzyme (a ribonuclease) that cleaves double-stranded RNA (dsRNA) and pre-micro RNA (miRNA) to form small interfering RNA (siRNA) molecules about 20–25 nucleotides long that serve as guide molecules for the degradation of mRNA molecules with sequences complementary to the siRNA.
RNA-Induced Silencing Complex (RISC complex)
Figure 17-25 Mechanisms of gene regulation by RNA gene silencing. In the cytoplasm, two systems operate to silence genes. (Middle) In siRNA mediated silencing, a precursor RNA molecule is processed by Dicer, a protein with RNAse activity to form an antisense single stranded RNA that combines with a protein complex with endonuclease activity. siRNA/RISC (RNA-induced silencing complex) binds to mRNAs with complementary sequences, and cuts the mRNA into fragments that are degraded. This process is called RNAi in animal cells, and posttranslational gene silencing (PTGS) in plants. (Right) A partially double-stranded precursor is processed by Dicer to yield microRNA (miRNA) that binds to complementary -untranslated regions (UTRs) of mRNA, inhibiting translation. In plants, miRNAs cause arrest of translation. (Left) Small RNAs, processed by Dicer, play a role in RNA-directed DNA methylation (RdDM). These RNAs combine with DNA methyl transferases (DMTases) to methylate cytosine residues in promoter regions (purple circles), silencing genes.
Small interfering RNAs and microRNAs are produced from double-stranded RNAs.
Small interfering RNAs and microRNAs are produced from double-stranded RNAs.
Yet Another Role for Dicer
RITS (RNA-induced initiation of transcriptional silencing) complex
In this pathway, short RNAs can also mediate RNA-directed DNA methylation (RdDM).
siRNAs:
Origin, Cleavage, Action, Target
Origin: mRNA, transposon, or virus
Cleavage of: RNA duplex or single-stranded RNA that forms long HairPins
Action: Some trigger degradation of mRNA, others inhibit transcription
Target: Genes from which they were subscribed.
miRNAs:
Origin, Cleavage, Action, Target
Origin: RNA transcribed from a distinct gene.
Cleavage of: single-stranded RNA that forms short HairPins
Action: Some trigger degradation of mRNA, others inhibit translation
Target: Genes other than those from which they were subscribed.
