chapter 14 - rna molecules & processing
3 areas/sequences where splicesome splices on intron
5' splice site (near 5' end) 3' splice site (near 3' end) branch point - adenine inside intron near 3' splice site
3' UTR
a sequence of nucleotides at the 3′ end of the mRNA that is not translated into a protein. However, it does affect the translation of the mRNA molecule as well as the stability of the mRNA
loss of 5' cap
prevents splicing of intron nearest to 5' cap will degrade in nucleus OR if transported into cytoplasm - ribosome won't recognize the mRNA and it degrades
alternative (exon) splicing
same pre-mRNA is spliced in more than one way to yield different proteins 95% of human genes under go this many more proteins created than protein encoding genes
promoter
the DNA sequence that the transcription apparatus recognizes and binds to initiate transcription. It is 5' or upstream to the start site for transcription (+1).
What are the three principal elements in mRNA sequences in bacterial cells?
(1) The 5′ untranslated region, which contains the Shine-Dalgarno sequence (2) The protein-encoding region, which begins with the start codon and ends with the stop codon (3) The 3′ untranslated region
How do the mRNAs of bacterial cells and the pre-mRNAs of eukaryotic cells differ? How do the mature mRNAs of bacterial and eukaryotic cells differ?
Bacterial mRNA is translated immediately upon being transcribed. Eukaryotic pre-mRNA must be processed and exported from the nucleus. Bacterial mRNA and eukaryotic pre-mRNA have similarities in structure. Each has a 5′ untranslated region as well as a 3′ untranslated region. Both also have protein-coding regions. However, the protein-coding region of the pre-mRNA is disrupted by introns. The eukaryotic pre-mRNA must be processed to produce the mature mRNA. Eukaryotic mRNA has a 5′ cap and a poly(A) tail, unlike bacterial mRNAs. Bacterial mRNA also contains the Shine-Dalgarno consensus sequence. Eukaryotic mRNA does not have the equivalent.
Suppose that a mutation occurs in the middle of a large intron of a gene encoding a protein. What will the most likely effect of the mutation be on the amino acid sequence of that protein? Explain your answer.
Because introns are removed prior to translation, an intron mutation would have little effect on a protein's amino acid sequence unless the mutation occurred within the 5′ splice site, the 3′ splice site, or the branch point and prevented removal of the intron from the pre-mRNA.
What is the function of the 5′ cap?
CAP binding proteins recognize the 5′ cap and stimulate binding of the ribosome to the 5′ cap and to the mRNA molecule. The 5′ cap may also increase mRNA stability in the cytoplasm. Finally, the 5′ cap is needed for efficient splicing of the intron that is nearest the 5′ end of the pre-mRNA molecule.
How is the poly(A) tail added to pre-mRNA? What is the purpose of the poly(A) tail?
Cleavage of the mRNA occurs 11-30 nucleotides downstream of the polyadenylation consensus sequence (AAUAAA). 50-250 adenines are then added to the 3' end of the RNA molecule. The presence of the poly(A) tail increases the stability of the mRNA molecule through the interaction of proteins at the poly(A) tail. The poly(A) tail also assists with the binding of the ribosome to the mRNA.
What is the concept of colinearity? In what way is this concept fulfilled in bacterial and eukaryotic cells?
Colinearity is the concept that the sequence of codons in the DNA of a gene has a direct correspondence with the sequence of amino acids in the protein. If we examine the location of three codons for three amino acids in a protein, the order of the codons in the gene is always the same as the order of the amino acids in the protein. The presence of large regions of noncoding DNA in introns means that DNA sequences that code for adjacent amino acids may be separated by many base pairs of DNA, but introns to not alter the order of codons in the gene.
Introns begin with ____ and end with ____.
GU and AG GU - 5' splice site /consensus seq. AG - 3' splice site / consensus seq.
How would the deletion of the Shine-Dalgarno sequence affect a bacterial mRNA?
In bacteria, the ribosome binds to the Shine-Dalgarno sequence to initiate translation. If the Shine-Dalgarno sequence is deleted, then translation initiation cannot take place, preventing protein synthesis.
What are some characteristics of introns?
Introns are intervening sequences that typically do not encode proteins. Eukaryotic genes commonly contain introns. However, introns are rare in bacterial genes. The number of introns found in an organism's genome is typically related to complexity— more complex organisms possess more introns.
Deletion of Poly(A) tail
Polyadenylation increases the stability of the mRNA and is required for translation. If eliminated from the premRNA, then the mRNA would be degraded quickly by exonucleases in the cytoplasm.
5. Summarize the different types of processing that can take place in pre-mRNA.
Several modifications to pre-mRNA take place to produce mature mRNA in eukaryotes. (1) Addition of the 5′ 7-methylguanosine cap to the 5′ end of the pre-mRNA (2) Cleavage at a site downstream of the AAUAAA consensus sequence at the 3′ end of the pre-mRNA (3) Addition of the poly(A) tail to the 3′ end of the mRNA immediately following cleavage (4) Removal of the introns (splicing)
Are the 5′ untranslated regions (5′ UTR) of eukaryotic mRNAs encoded by sequences in the promoter, exons, or introns of the gene? Explain your answer.
The 5' UTR is located in the first exon of the gene. It is not part of the promoter because promoters for RNA polymerase II (which transcribes pre-mRNA) are not normally transcribed, and the 5' UTR is in the mRNA. It is not located in an intron because introns are removed during processing of pre-mRNA, and the 5' UTR is part of the mRNA.
In eukaryotes, what is the 5′ cap?
The 5′ end of eukaryotic mRNA is modified by the addition of the 7-methyl guanine 5′ cap. The cap consists of an extra guanine nucleotide linked 5′ to 5′ to the mRNA molecule. This nucleotide is methylated at position 7 of the base. The ribose sugars of adjacent bases may be methylated at the 2′ -OH.
In bacteria, what is the function of the Shine-Dalgarno consensus sequence?
The Shine-Dalgarno consensus sequence functions as the ribosome-binding site on the mRNA molecule.
Duchenne muscular dystrophy is caused by a mutation in a gene that comprises 2.5 million base pairs and specifies a protein called dystrophin. However, less than 1% of the gene actually encodes the amino acids in the dystrophin protein. On the basis of what you now know about gene structure and RNA processing in eukaryotic cells, provide a possible explanation for the large size of the dystrophin gene.
The large size of the dystrophin gene is due to the presence of many intervening sequences or introns within the coding region of the gene. Excision of the introns through RNA splicing yields the mature mRNA that encodes the dystrophin protein.
A geneticist discovers that two different proteins are encoded by the same gene. One protein has 56 amino acids and the other 82 amino acids. Provide a possible explanation for how the same gene could encode both of these proteins.
The pre-mRNA molecules transcribed from the gene are likely processed by alternative processing pathways. Two possible mechanisms that could have produced the two different proteins from the same pre-mRNA are alternative splicing or multiple 3′ cleavage sites in the pre-mRNA. The cleavage of the pre-mRNA molecule at different 3′ cleavage sites would produce alternatively processed mRNA molecules that differ in size. Translation from each of the alternative mRNAs could produce proteins containing different numbers of amino acids. Alternative splicing of the pre-mRNA could produce different mature mRNAs, each containing a different number of exons and thus the mRNAs differ in size. Again, translation from each alternatively spliced mRNA would generate proteins that differ in the number of amino acids contained.
A geneticist induces a mutation in a cell line growing in the laboratory. The mutation occurs in a gene that encodes a protein that participates in the cleavage and polyadenylation of eukaryotic mRNA. What will be the immediate effect of this mutation on RNA molecules in the cultured cells?
The protein is needed as part of the process for cleavage of the 3′ UTR and for polyadenylation. A nonfunctional protein would result in mRNA lacking a poly(A) tail, and the mRNA would not be translated and would be degraded more quickly in the cytoplasm by nucleases.
What is a spliceosome and what does it do?
The spliceosome consists of small RNA molecules and a large number of proteins. Splicing of pre-mRNA nuclear introns occurs through the action of the spliceosome which recognizes consensus sequences at the 5' splice site, the 3' splice site and the branch point.
poly (a) tail
added to the 3' end of the pre-mRNA. It affects mRNA stability and translation.
in bacteria/prokaryotes, transcription & translation are
coupled - happens together
colinear
direct correspondence between the nucleotide sequence of DNA and the amino acid sequence of a protein (discovered by Crick) # of nucleotides in gene = # of amino acids in protein of gene (1:1 ratio) eukaryotic genes have stretches of coding & non-coding sequences - not colinear
5' cap
functions in the initiation of translation and mRNA stability
alternative promoters/alternative 5' UTRs
gene w more than 1 promoter = multiple RNAs with different 5' UTRs dif 5' UTRs = dif stability or regulation
5' cap & Kozak consensus
in eukaryotes, there is no shine-dalgarno sequence before start codon 5' cap at 5' end is where ribosome binds to until if finds AUG (start codon) in the Kozak consensus Kozak - surrounds the start codon
polyadenylation consensus sequence
lies near the 3′ end of the pre-mRNA. It determines the location of the 3′ cleavage and poly(A) tail addition to the pre-mRNA molecule.
5' UTR
lies upstream of the translation start site The eukaryotic ribosome binds at the 5′ cap of the mRNA molecule and scans to the first methionine codon (AUG) in a Kozak consensus. The region 5′ of this start codon is the 5′ UTR.
introns not removed
mRNA cannot leave the nucleus
example of when alternative splicing & multiple 3' cleavage sites occur in the same pre-mRNA
mammalian gene that encodes calcitonin
deletion of polyadenylation signal (AAUAAA) consensus sequence
no cleavage or polyadenylation of pre-mRNA affects stability and translation of the mRNA unstable & degrades before translation
introns
non-coding sequence removed DURING RNA processing all eukaryotic genes have this (rare in bacteria) # ranges by complexity; more complex = more introns
exons
nucleotide sequences in DNA retained AFTER non-coding sequence is removed include 5' UTR, coding regions & 3' UTR
transcription start site
the location of the first transcribed nucleotide of the mRNA and is located 25 to 30 nucleotides downstream of the TATA box.
in eukaryotes, transcription & translation are
uncoupled - happens separately
pre-mRNA
when eukaryotic RNA polymerases transcribe the DNA of a gene the RNA produced is called a pre-mRNA the pre-mRNA contains introns and 3' material that will be cleaved off the RNA molecule
mature mRNA
when the introns have been removed and modifications have been made to the 5' and 3' ends the mRNA is referred to as a mature mRNA the mature mRNA is ready for translation
alternative 3' cleavage sites
where 2+ potential sites for cleavage & polyadenalation are present in pre-mRNA generates mRNAs with different 3' UTRs = different stabilities = different amount of protein production 50% of human genes have this
