Unit 7 Flashcards
Describe the function of rRNA.
Ribsomal RNA. Components of ribosomes help carry out protein synthesis:
- Bind Shine-Dalgarno
- Make up active site and most of whole complex
Describe the function of mRNA.
Messenger RNA strand made from coding DNA template. Transfers stored genetic information as transcription to be translated into protein.
Describe the function of tRNA.
Transfer RNA: Adapter RNAs that translate the codons of mRNA into appropriate amino acids linked on the polypeptide.
Compare the pentose ring of DNA and RNA,.
DNA pentose is deoxygenated or has an H at 2’ C where the ribose of RNA has an OH and an H.
Which nitrogen bases (C,A,G,U, and/or T) are found in DNA? In RNA?
Typically, DNA has A, T, G, and C.
Typically, RNA has A, U, G, and C. However, the pentose structure - not the bps - is what actually designates molecule as DNA or RNA.
Distinguish between monocistronic and polycistronic mRNA. Is eukaryotic mRNA monocistronic or polycistronic? What about prokaryotic mRNA?
Monocistronic - mRNA codes for only one polypeptide product. (most eukaryotic mRNA)
Polycistronic - mRNA codes for two or more different polypeptides. (most prokaryotic mRNA)
Is DNA usually double or single stranded?
Double.
Is RNA usually double or single stranded? )? Does single stranded RNA have any double stranded regions?
Single stranded. Folds on to itself or finds self complementary regions, where AU and GC pair (+ irregular G-U pair). Exists as right handed single strand helices (base stacking interactions) and forms hairpin duplexes as most common secondary (hydrogen bonding driven) structure.
Give an overview of transcription by RNA polymerase.
Essentially identical to mechanism by DNAp:
- Uses Mg 2+ and Asp to selectively stabilize and destabilize triphosphate group and 3’ OH of terminal
- Deprotonates and allows nucleophilic attack of phosphate
- Breaks phosphoanhydride bond and forms stabilized, phosphodiester bond product
Distinguish between RNA polymerases and DNA polymerase I in terms of substrates.
DNAp - deoxynucleoside triphosphate (dNTPs) substrates
RNAp - nucleoside-triphosphate substrates (NTPs)
Distinguish between RNA polymerases and DNA polymerase I in terms of whether a primer is needed.
DNAp - needs primer (RNA made by primase)
RNAp - does not need primer (binds to and starts synthesis from a promoter)
Distinguish between RNA polymerases and DNA polymerase I in terms of direction of synthesis.
DNAp - add 5’ to 3’
RNAp - also adds 5’ to 3’
Distinguish between RNA polymerases and DNA polymerase I in terms of what nucleophilic attack occurs during bond formation.
DNAp - deprotonated 3’ OH attacks alpha phosphate of incoming dNTP
RNAp - deprotonated 3’ OH attacks alpha phosphate of incoming NTP
Distinguish between RNA polymerases and DNA polymerase I in terms of the role of pyrophosphate and pyrophosphatase in the overall reaction.
DNAp - PPi breakdown by pyrophosphatases drives rxn forward both with Le Chat and thermodynamically.
RNAp - PPi breakdown does not occur as driving mechanism. First added nucleotide retains PPPi group.
Distinguish between RNA polymerases and DNA polymerase I in terms of the requirement of a template.
DNAp - uses either and both strands of DNA as identical templates.
RNAp - uses only one strand of DNA - the template strand (rather than non-template)
What is meant by the terms “template” and “non-template” strands with regards to RNA polymerase synthesis?
Template = strand which mRNA is synthesized from. Non-template is DNA analog of mRNA.
Does the same strand of the chromosome always serve as the template strand?
No.
Discuss the quaternary structure of Taq RNA polymerase.
Multiple subunits - beta, beta prime, alpha 2x, and omega. Sigma subunit binds as a specificity factor.
Does RNA polymerase have 3’ to 5’ exonuclease activity? Why is it unnecessary?
No.
1) Many mRNAs produced
2) Degraded regularly
Discuss the general organization of an E. coli promoter.
Site recognized by sigma 70. Has consensus sequences around -10 and -35. Some promoters also have a UP (upstream promoter) sequence that is further back (more negative).
What is meant by the term “consensus sequence”?
The sequence that is made up of the most commonly found base at each site in the sequence - theoretical, however, and not necessarily a real sequence possessed by any organism.
Define the numbering system which is used to identify regions such as the -10 and -35 regions.
0 refers to the start site of transcription. Positions before this site are -# and after are +#.
What are promoters?
Sequence where RNAp binds to the DNA, but not necessarily where it begins transcription. Promoter sequences can exist before or after site of transcription initiation.
What is the significance of the -35 and -10 regions around an E.
These are consensus sequence sites that direct binding of RNAp with the sigma factor.
What is the relationship between the “strength” of a promoter (i.e. the frequency at which transcription is initiated at a particular promoter) and the degree of match to a consensus sequence?
Closer to consensus sequence = stronger promoter
What is the function of the sigma (σ) subunit?
Specificity factor for RNAp. Localizes RNAp to promoter sites.
Discuss the events in transcription initiation.
1) Sigma factor and RNAp associate
2) Binding of holoenzyme to promoter = closed complex binding
3) RNAp opens strands and begins transcription = open complex and initiation
4) promoter clearance and dissociation of sigma factor
Is transcription usually regulated at the level of initiation, elongation, or termination? Is it ever regulated at the other levels?
Regulated at initiation mostly. Can be regulated at any of these levels.
Discuss two distinguishing features of the termination signal in the mRNA in rho independent termination of transcription in prokaryotes.
1) Palindromic sequence shortly before termination site - forms mRNA hairpin.
2) AAA sequence in template - gives UUU sequence in mRNA that binds and causes kinetic pause to allow for hairpin formation and detachment of mRNA.
Describe the sequences typically found at a eukaryotic promoter. How do these sequences compare to those found at a prokaryotic promoter?
1) TATA box before initiation site
2) Initiator sequence (Inr) right after initiation site
3) Can have many different up stream regulatory sequences
What is a transcription factor?
A protein that binds to regulatory sequence and regulates transcription initiation.
Discuss the formation of a primary transcript and its processing to an mRNA.
Primary transcript hot off the DNA.
mRNA is post processed by:
1) 5’ G cap assembly
2) Cleavage of non coding end sequence and addition of Poly A tail at 3’ end (polyadenylation)
3) RNA splicing (to remove introns) by snRNAs (small nuclear RNAs) for spliceosomal introns and by themselves (lariat formation) for self splicing
Describe and list two functions of the cap.
1) Protect end from degradation
2) Help mRNA localize to ribosome.
Do all eukaryotic genes contain introns? Do most?
No (not histone coding genes) and yes.
Does splicing take place at the level of DNA or RNA?
RNA.
Point out features at exon-intron junctions that provide signals for correct splicing. What are the consequences of imprecise splicing?
Key signal sequence (5’ GU —- AG 3’) attracts U1 (snRNA) binding and induces spliceosomal splicing.
How do individual snRNAs locate the consensus sequences that flank introns?
It is complementary to an snRNA sequence.
Point out the location and identity of the nucleotide that will make the initial attack on the 3’ end of the exon in self splicing introns.
Nucleophilic adenine inside the intron makes the initial attack on the 3’ end of the last intron nucleotide.
What is the “lariat structure”?
A looped structure formed by the attack of the internal adenine on the end of the intron and resulting self splicing.
What is the function of the poly (A) tail?
1) Protect mRNA from degradation
2) Serve as binding site for various proteins.
Discuss the key steps in the addition of a poly (A) tail.
1) Is the tail encoded in the gene?
2) Name the enzymes involved in making the poly (A) tail.
3) Does this enzyme add residues in a template directed manner like DNA polymerase?
1) No, there is a cleavage site sequence located in a non coding region at the 3’ end, however. AAUAAA sequence right before cleavage site and G and U rich sequence after.
2) Polyadenylate polymerase
3) No, no template required.
Distinguish between mRNA post transcriptional processing and “RNA editing.”
Modification by alteration of sequence vs RNA editing by directly altering the bases.
Distinguish between poly A site choice and alternative splicing.
1) Alternative splicing - different exons may be removed in addition to introns so the ligated products give splice variants.
2) Alternative cleavage - multiple possible cleavage sites means longer or shorter mRNA products depending on where the polyadenylation takes place
