Chapter 12: Synthesis and Processing of Bacterial RNAs Flashcards
Module 10
Bacterial mRNA can immediately be translated, however ______ and _____, on the other hand, are first transcribed as pre-RNAs which become functional only after a series of cutting events and chemical modifications
- tRNAs
- rRNAs
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rRNA processing
- Bacteria synthesize three different rRNAs, called
- the names indicating the sizes of the molecules as measured by _____ _____
- three genes for these rRNAs are linked into a single ______ ______, usually present in multiple copies
- pre-rRNA contains copies of all three rRNAs
- Cutting is needed to release the mature rRNAs. These cuts are made by
- 5S rRNA, 16S rRNA, and 23S rRNA
- sedimentation analysis
- transcription unit
- ribonucleases III, P, and F
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rRNA processing
steps
- cuts are made by
- ribonucleases III (endonucleases)
- P (ribozyme) and F
- found in all organisms
- cut ends are then trimmed by the exonuclease activities of ribonucleases M16, M23, and M5 (exonucleases) to produce mature rRNAs
tRNA Processing
Genes for tRNAs are distributed around bacterial genomes, some on their own and some as _____ _____ in multi-tRNA transcription units. In some bacteria, they occur as ______ in the rRNA transcription units, as is the case with E. coli, which has either one or two tRNA genes between _____ _____. All pre-tRNAs are processed in similar though not identical ways
- tandem arrays
- infiltrators
- mRNA genes
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tRNA Processing
steps
- tRNA sequence within pre-tRNA adopts its cloverleaf structure
- two additional hairpin structures form, one on either side of the tRNA
- ribonuclease E or F cuts just upstream of one of the hairpins, forming a new 3’ end
- Ribonuclease D (exonuclease) trims seven nucleotides from this new 3’ end and pauses, waiting for ribonuclease P
- ribonuclease P makes a cut at the start of the cloverleaf, forming the 5’ end of the mature tRNA
- Ribonuclease D then removes two more nucleotides, creating the 3’ end of the mature molecule
- mature tRNAs must end with the trinucleotide 5¢–CCA–3¢.
- in some pre-tRNAs this sequence is absent, or is removed by the processing ribonucleases
- removal occurs when 3’ ends are created by an endonuclease called ribonuclease Z
- makes a cut adjacent to the first base pair in the tRNA cloverleaf removing the region containing terminal CCA
- When CCA is absent, it’s added by one or more template-independent RNA polymerases such as tRNA nucleotidyltransferase
one of ribonuclease P’s subunits is made of ____ rather than protein. these _____ subunits are present in several enzymes involved in RNA processing, including ribonuclease _____, which works with ribonuclease P in processing of eukaryotic pre-rRNAs. These hybrid _____-_____ enzymes may be relics of the _____ world
- RNA
- RNA
- MRP
- protein–RNA
- RNA
Module 10
The final type of processing that occurs with pre-RNAs is the chemical modification of ______ within the transcript. This occurs with _____ and ______. A broad spectrum of chemical changes has been identified with different pre-RNAs: over 50 different modifications are known in total. Most of these are carried out directly on an existing nucleotide within the transcript but two modified nucleotides, ______ and ______, are put in place by cutting out an entire nucleotide and replacing it with the modified version
- nucleotides
- prerRNAs
- pre-tRNAs
- queosine
- wyosine
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Many of these unusual nucleotides were first identified in tRNAs, where one in ten nucleotides becomes altered. Reasons why these modifications may occur are
- may mediate the recognition of individual tRNAs by the enzymes that attach amino acids to these molecules
- increases the range of the interactions that can occur between tRNAs and codons during translation
- enabling a single tRNA to recognize more than one codon
Ribosomal RNAs are modified in two ways:
- addition of methyl groups to, mainly, the 2’–OH group on nucleotide sugars, and by conversion of uridine to pseudouridine
- occurs at the same position on all copies of an rRNA
- same in different species
- bacterial rRNAs are less heavily modified than eukaryotic ones
- function behind modifications have not been identified
- most occur within those parts of rRNAs thought to be most critical to the activity of these molecules
- Often two or more nucleotides in the same region are modified at once
Module 10
RNA degradation is equally important, especially with regard to ______ whose presence or absence in the cell determines which proteins will be ______. Degradation of specific mRNAs could be a powerful way of ______ genome expression. rate of degradation of an mRNA can be estimated by determining its ______ in the cell. There are considerable variations between/within organisms. Bacterial mRNAs are generally turned over very rapidly, their half-lives rarely being longer than a ______ minutes while Eukaryotic mRNAs are longer lived, with half-lives of, on average, _______ minutes for yeast and several ______ for mammals
- mRNAs
- synthesized
- regulating
- halflife
- few
- 10–20
- hours
Module 10
Bacterial mRNAs are degraded in the ______ direction
3’→5’
Module 10
RNA-degrading enzymes that are thought to be involved in mRNA degradation
- RNase E and RNase III
- endonucleases that make internal cuts in RNA molecules.
- RNase II
- exonuclease that removes nucleotides in the 3’→5’ direction
- Polynucleotide phosphorylase (PNPase)
- exonuclease that removes nucleotides in the 3’→5’ direction
- requires inorganic phosphate as a cosubstrate
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RNA-degrading process
- most mRNAs have a hairpin structure near the 3’ end
- hairpin that induced termination of transcription
- The termination hairpin blocks the exonuclease activities of RNase II and PNPase
- termination hairpin is removed by endonuclease action of RNase E and/or RNase III
- removal exposes a new end for RNase II and PNPase to attach and destroy the functional activity of the mRNA
degradosome
- contains RNase E and PNPase
- include an RNA helicase
- might be involved in both rRNA and mRNA degradation.
- not clear and a few researchers are sceptical about its actual existence,
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Synthesis and Processing of Eukaryotic RNA
promoter clearance
transition from the preinitiation complex to a complex that has begun to synthesize RNA
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Synthesis and Processing of Eukaryotic RNA
Capping of RNA polymerase II transcripts
- occurs immediately after initiation
- Successful promoter escape could be linked with capping
- completed before the transcript reaches 30 nucleotides
- an extra guanosine is added to the extreme 5’ end of the RNA
- involves a reaction between the 5’ triphosphate of the terminal nucleotide and the triphosphate of a GTP nucleotide
- the outermost phosphate is removed, as are the β and γ phosphates of the GTP, resulting in a 5’–5’ bond
- carried out by the enzyme guanylyl transferase
- conversion of new terminal guanosine into 7-methylguanosine by attachment of a methyl group to nitrogen number 7 of the purine ring
- catalyzed by guanine methyltransferase
- called a type 0 cap
- commonest form in yeast
- In higher eukaryotes, additional modifications occur
- A second methylation replaces the hydrogen of the 2’–OH group of what is now the second nucleotide in the transcript. This results in a type 1 cap.
- If this second nucleotide is an adenosine, then the amino group attached to carbon number 6 of the purine ring might also be methylated.
- Another 2’–OH methylation might occur at the third nucleotide position, resulting in a type 2 cap.
- two capping enzymes make attachments with the CTD
- possible that they are intrinsic components of the RNA polymerase II complex during promoter clearance
Module 10
GU–AG introns
- first two nucleotides of the intron sequence are 5’–GU–3’ and the last two are 5’–AG–3’
- all members of this class are spliced in the same way
- conserved motifs
- recognition sequences for RNA-binding proteins involved in splicing or playing some other central role in the process
- parts of longer consensus sequences that span the 5¢ and 3¢ splice sites
- vary in different types of eukaryote
- can be described as
- 5’ splice site/donor site:
- 5’–AG↓GUAAGU–3’
- 3’ splice site/acceptor site:
- 5’–PyPyPyPyPyPyNCAG↓–3’
- Py = two pyrimidine nucleotides (U or C)
- N = any nucleotide
- 5’ splice site/donor site:
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GU–AG introns
splicing pathway for GU–AG introns
- Cleavage of the 5’ splice site
- hydroxyl attack (transesterification reaction) between the OH attached to the 2’ carbon of an adenosine nucleotide located within the intron sequence
- In yeast, this adenosine is the last one in the conserved 5’–UACUAAC–3’ sequence
- results in cleavage of the phosphodiester bond at the 5’ splice site. this exon is now free.
- the newly half-released intron creates a new 5’–2’ phosphodiester bond by linking the first nucleotide (the G of the 5’–GU–3’ motif) with the internal adenosine (the one that attcked) causing it to loop back on itself to create a lariat structure.
- hydroxyl attack (transesterification reaction) between the OH attached to the 2’ carbon of an adenosine nucleotide located within the intron sequence
- Cleavage of the 3’ splice site
- second transesterification reaction happens, by the 3’–OH group at the end of the upstream exon (the one released)
- It attacks the phosphodiester bond at the 3’ splice site, cleaving and releasing the intron/lariat structure
- introng/lariat is converted back to a linear RNA and degraded
- 3’ end of the upstream exon joins to the newly formed 5’ end of the downstream exon, completing the splicing process
Module 10
GU–AG introns
splicing pathway difficulties lies with topological problems. explain
- substantial distance that might lie between splice sites, possibly a few tens of kilobases
- means needed of bringing the splice sites into proximity
- selection of the correct splice site
- All splice sites are similar
- wrong splice sites could be joined, resulting in exon skipping— the loss of an exon from the mature mRNA
- site within an intron or exon that has sequence similarity with the consensus motifs of real splice sites can result in cryptic splice site
- these sites are present in most pre-mRNAs and must be ignored by the splicing apparatus
Module 10
GU–AG introns
central components of the splicing apparatus
- snRNAs
- U1, U2, U4, U5, and U6
- short molecules (between 106 nucleotides [U6] and 185 nucleotides [U2] in vertebrates)
- associate with proteins to form small nuclear ribonucleoproteins, snRNPs
- snRNPs
- together with other accessory proteins, attach to the transcript and form a spliceosome
- spliceosome
- structure where splicing reactions occur
