Biomg 3300 Unit 7 Quiz

Function of rRNA?

class of RNA components on ribosomes

Function of mRNA?

class of RNA molecules translated by ribosomes to produce proteins

Function of tRNA?

combines covalently with amino acids as the first step in protein synthesis, translates info in mRNA into specific sequence of amino acids

Identity of pentose ring (ribose vs deoxyribose)

DNA: H at 2' carbon

RNA: OH at 2' carbon

Which nitrogen bases are found in DNA? RNA?

DNA: A, C, T, G

RNA: A, C, G, U

Distinguish between monocistronic and polycistronic mRNA? Is eukaryotic mRNA mono or poly? What about prokaryotic?

Mono: mRNA that can be translated into one protein

Poly: mRNA that can be translated into 2 or more different polypeptides

eu: mono

pro: poly

Is DNA usually single or double? RNA?

DNA: double

RNA: single

Does single stranded RNA have any double stranded regions? Point out a hairpin loop (what is it also called), Define 2 types of base pairs

yes: when 2 strands of complementary RNA are paired --> forms A form right handed double helix or when RNA folds onitself

Breaks caused by mismatch loops --> bulge and internal loop

Hairpin: forms between self complementary sequences (RNA folds on itself) --> this can happen bc RNA is single stranded

A and U

G and C

Note RNA structures in tert structure

ok and ask

just know it forms tert structure

What does a double helix RNA have that is not similar to mRNA?

much more secondary structure

What is less stable DNA or RNA?

RNA

What is the substrate for RNA polymerase vs DNA polymerase?

RNA: NTP

DNA: dNTP

Is a primer needed in RNA poly or DNA poly?

RNA: no

DNA: yes

What is the direction of synthesis in RNA poly and DNA poly?

5 to 3

What is the nucleophilic attack in bond formation in both?

3'-OH acts as a nucleophile and attacks the a phosphate of the incoming NTP (or dNTP) --> this causes the phosphate to be released

What is the role of pyrophosphate and pyrophosphatase in overall rxn?

Hydrolysis of Ppi drives the overall rxn towards products

Is there a requirement for a template for both?

yes, they both need a DNA template

Draw out the overview of transcription by RNA polymerase

ok

O's of phosphate stabilized by Mg2+ which is stabilized by asp residues of RNA polymerase

What is meant by the terms template and non template strands?

nontemplate (coding) is complemenatry to the template

strand that serves as a template for RNA synthesis (template strand)

Does the same strand of the chromosome always serve as template strand?

non template (coding) is usually 5 to 3

template is usually 3 to 5

discuss the quaternary structure of RNA polymerase.

Has 5 core subunits (a, a, B, B', and w)

crab claw shape is done by the beta subunits

6th sigma unit is transiently (not always) bound to the top of the beta subunits and directs the enzyme to specific binding sites on the DNA (sigma subunit binds to the promoter sequences)

When we are talking about the sigma subunit, are we talking about pro or eu?

pro

Does RNA polymerase have 3' to 5' exonuclease activity?

No, unnecessary bc many copies of RNA are generally produced by a single gene and all RNA's are eventually replaced so there is no need for proofreading

What is meant by the term consensus sequence? What is this in?

this is in E. coli

- certain nucleotides that are common at each position forms a consensus sequence

- determined through analysis and comparison of most common class of bacterial promoters

- recognized by RNA polymerase as binding site

Define the numbering system which is used to identify the regions such as -10 and -35?

DNA base pairs that correspond to the beginning of the RNA molecule are given + numbers, and those preceeding the RNA start site are given - numbers

1st base copied is +1

-10: DNA sequence located 10 BP before transcription start site

What is the significance of -10 and -35 regions?

2 consensus sequences at those regions are recognized by RNA polymerase as binding site --> sigma subunit is thought to bind here

What is the relationship to the strength of a promoter and the degree of match to the consensu sequence?

closer the promoter is to the consensus sequence --> better RNA polyermase will bind and faster the rate of transcription will be

What is the function of the sigma subunit?

binds transiently to the core and directs the enzyme to specific binding sites on DNA

Discuss the events in transcription and elongation?

2 major parts: binding and initiation

1) RNA polymerase binds (+1 position) and is directed by sigma factor

2) transcription bubble opens and DNA is opened up

3) transcription is initiated and RNA is elongated

4) promoter is cleared after elongation

5) elongation continues, sigma is replaced with NusA

6) transcription is terminated, NusA disassociates and RNA polymerase is recycled

Is transcription usually regulated at the level of intiiation, elongation, or termination? Is it ever regulated at other levels?

initiation: it can occur at any step including elongation and termination but don't want to make unecessary mRNA bc it wastes energy

Discuss 2 distinguishing features of termination signal in prokaryotes?

1) RNA hairpin forms due to palindromic structure

2) Highly conserved A and U residues at 3' end (weakly bond due to 2 H bonds), Formation of the hairpin structure disrupts the AU base pairs which allows the mRNA to disassociate

What is eukaryotic machinery like?

machinery in nucleus, 3 RNA polymerases, RNA poly 2 is most common for mRNA

Describe the sequences usually found at eukaryotic promoter?

1) TATA box at -30: major assembly pt for proteins of preinitiation complexes of Poly II

2) site where DNA is unwound (transcription starts here) this is at +1

How do these sequences compare to those found at prokaryotic promoter?

Eu promoter sequences are more variable than pro

majority of eu lacks TATA box, consensus Inr element, or BOTH

What is a transcription factor?

in eukaryotes, protein that affects regulation and transcription of a gene--> binds to regulatory sequence near or within the gene and interacts with RNA polymerase or other transcription factors

Discuss the formation of a primary transcript and its processing to mRNA?

A primary transcript is a newly synthesized RNA molecule

Addition of 5' cap, removal of noncoding intron sequences, transcript cleavage, polyadenylation (3' poly A tail)

Describe and list 2 functions of the cap

5' cap is 7-methylguanosine, it is linked to the 5' terminal cap of the mRNA through 5',5'-triphosphate linkage

1) cap protects mRNA from degradation by other enzymes and ribonucleases

2) participates in binding of mRNA on ribosome to initiate translation

Distinguish between exons and introns?

exons: segment of mRNA that remains after post transcriptional processing and is transcribed into protein

introns: seq of nucleotides that is transcribed but excised before the gene is translated

Do all eukaryotic genes contain introns?

the vast majority of genes in vertebrates contain introns (among the few exceptions are those that encode histones)

Does RNA splicing occur at DNA or RNA?

RNA

Point out features at exon-intron junctions of group II introns that provide signals for correct splicing

dinucleotide sequence of GU at 5' end and AG at 3' end (of INTRON)

- 2' hydroxyl of A residue within the intron attacks the phosphate between the exon and intron

- Then the 3' hydroxyl of the exon that is displaced acts as a nucleophile and attacks 3' end of intron

What is a spliceosome? What is a snRNP (also called a snurp)?

Spliceosome: complex of RNA and proteins involved in splciing in mRNA in eu cells

snRNP: small nuclear ribonucleoproteins, specialized RNA-protein complexes that make up spliceosomes. Each snRNP contains one class of eukaryotic RNA's known as small nuclear RNA's (snRNA)

How do individual snRNA's locate the consensus sequences that flank introns?

U1 snRNA contains a sequence complementary to the 5' splice site (GU) and U1 snRNP binds to this region by forming a RNA duplex with pre-mRNA. A U2 snRNP binds to the 3' end (AG) also by base pairing, which then identifies the adenylate that becomes the nucleophile

What is the lariat structure

structure formed when intron folds in on itself

What is the function of the poly A tail?

-binding site for one or more proteins

- variety of roles in coordinating transcription/translation

- protects mRNA from enzymatic destruction

is the poly A tail encoded in the gene?

No, it contains no template

Name the enzymes involved in making the poly A tail?

polyadenylate polymerase

- endonuclease (clevaes the mRNA)

Does this enzyme require residues in a template directed manner like DNA poly?

No, the transcript is extended beyond the site where the poly A tail is added, then is cleaved at the poly A additional site by an endonuclease. DOES require cleaved mRNA as primer

Distinguish between RNA editing and splicing?

RNA editing: processes that adds/deletes bases in the coding regions of primary transcripts. Occurs post translational on mature mRNA

splicing: happens on post transcriptional primary mRNA

splicing is the removal of whole segments of nucleotides (introns) while RNA editing is the removal/addition of individual bases

Distinguish between poly A site choice and alternative splicing. Discuss the roles of each of these mechanisms in generating protein diversity from a single gene

Poly A: strand cleavage and add. of 3' poly A tail at alternative locations within a mRNA transcript to generate different mature RNA's

Alt splicing: process in which nonconsecutive exons are selectively spliced in alt ways to generate diff mature RNA's

What is meant by RNA world? What evidence is used?

RNA was the first genetic material bc it could store info and has catalytic activity and replicate itself

What evidence is used for support of the idea of an RNA world?

1) prebiotic chemistry experiments --> invitro experiments showed that RNA can perform functions to support an RNA world

2) existence of catalytic RNA

3) catalytic repertoire of ribozymes: ribosomes are RNA enzymes that catalyze chemical rxns

4) structure of ribosomes: ribosomes are proteins but they have an RNA component

5) extant vestiges of an RNA world: features in organisms that would not exist if RNA was not the first genetic material

6) progress in search of an RNA replicator: trying to find a ribozyme/ribozyme system where a ribosome brings about its own synthesis in a template directed manner

What are 3 mechanisms which control the activity of enzymes in the cell?

1) allosteric modification

2) covalent modification

3) proteloytic cleavage: zymogen is cleaved to form active enzyme

Distinguish between constitutive and regulated gene expression?

constitutive: gene for products is required at all times and expressed a more/less constant level in every cell

regulated: when expression of a gene changes in response to rise/fall of cellular levels of molecular signals

Define specificity factor?

a protein that alters the specfity of RNA polymerase for a given promoter

Define repressor

protein that binds to regulatory sequence/operator for a gene blocking its transcription

Define activator

protein that enhances RNA polymerase/promoter interaction

Define operator

region of DNA that interacts with a repressor protein to control expression of a gene

Define negative and pos regulation

neg: regulation by means of a repressor protein that blocks transcription

pos: regulation by means of activator protein that enhances acticity of RNA poly at promoter

Describe the key features of operons? Where are they found? Is this monocistronic or polycistronic mRNA?

Activator, Promoter, Operator (repressor binding site)

Operator and Activator are regulatory binding sites

Gene cluster

found in pro

polycistronic (many genes on one mRNA)

Write the equation representing the hydrolysis of lactose? What is the name of the enzyme that catalyzes this rxn? What is the side product?

lactose + H20 --> glucose and galactose

Enzyme: B- galactosidase

Side: allolactose (isomer)

Draw a diagram of the lac operon?

P=

O1=

o2=

Structural genes

whole thing is operon?

p=promoter

O1: main operator for lac operon

O2 and O3: secondary operator site of lesser affinity for lac repressor

Lac Z, Y, A: structural genes

Name each of the 3 structural genes and describe the function for each?

Lac Z: encodes B-galactosidase (hydrolyszes the glycosidic bond between monosaccharides of lactose)

Lac Y: encodes galactosidase permease: allows lactose to move across the plasma membrane into the cytoplasm

Lac A: Encodes thiogalactoside transacetylase: gets rid of toxic byproducts of the rxn

Describe how the lac operon can be negatively regulated? What protein is involved? Name the gene that encodes this protein? What molecule acts as the inducer?

Lac repressor (encoded by the LacI gene)

Allolactose is the inducer: causes the repressor to release from the operator

How do proteins interact at specific DNA sequences? What type of interaction is involved? Within the DNA double helix, where does this interaction occur?

AA side chains of (Asn, Gln, Glu, Lys, Arg) hydrogen bond to bases in DNA

mainly H bonds

occurs on the major groove of the DNA

Describe the phenomenon called catabolite repression

Restriction of expression of genes required for catabolism of lactose and other sugars in presence of glucose, even when these secondary sugars are also present

--> idea that we want to use glucose as our main energy source to optimize growth

Discuss the role of cAMP receptor protein (CRP) and cAMP in the role of pos regulation of the lac operon?

CRP binds to cAMP --> cAMP is signaled when there is low glucose

cAMP and CRP--> together this increases RNA polymerase for the promoter which increases transcription

High glucose and high lactose?

high lactose means allolactose is present

no CRP+ cAMP

constitutive expression

Low glucose and high lactose?

high transcription

low lactose and high glucose?

no expression

Low glucose and low lactose?

no expression

--> no matter what, if you have low lactose there is no expression

List 5 important features that distinguish regulation of gene expression in eukaryotes from prokaryotes?

1) access to eukaryotic promoters is restricted by structure of chromatin (condensed)

2) + mechanisms are more prominant in eukaryotic regualtion

3) regulatory mechanisms involve lnRNAs (long coding RNAs)

4) eu has larger more complex multimeric protein complexes

5) transcription in eukaryotic nucleus is separated from translation in the cytoplasm

Distinguish between heterochromatin and euchromatin?

heterochromatin: condensed form of chromatin that is transcriptionally inactive and is generally associated with particular chromosome structures

euchromatin: less condensed form of chromatin that is transcriptionally active

Discuss the structural features of active chromatin (open or condensed)

1) position of nucleosome (less tighly packed)

2) presence of histone variants

3) covalent modifications of nucleosomes (methylation or acetylation)

What is meant by chromatin remodeling?

alteration of chromatin structure from condensed to open

What is an enhancer?

segment of DNA that faciliates expression of gene, located upstream from promoter

What are basal transcription factors used for?

assembles with RNA poly II at TATA box (TBP)

- this is a protein

- directly interacts with DNA

What are transcription activators?

binds to enhancer and facilitates transcription

What are chromatin modification and remodelling proteins?

need proteins that will make DNA accessible

- nucleosome positioning, covalently modify nucleosomes, histone changes

What do coactivators do?

-this does not directly interact with DNA

- mediates interactions between transcription activators (that may be bound distances away) and events at the TATA box with the promoter

What do architectural regulators do?

facilitates DNA looping

Discuss riboswitches?

binding of ligand to aptamer (a part of the mRNA that can fold into a 3D structure) leads to stabilization of transcription terminator, blockage of ribosome-binding site (blocks translation), and regulation of intron splicing (in fungi and plants)

this is cis regulation bc the mRNA is regulating itself

Discuss the synthesis and processing of miRNA's, what do they do?

target mRNA for hydrolysis

1) pri-mRNA is transcribed by RNA poly 2

2) pri-mRNA is reduced to pre-miRNA by Drosha and DGCR8

3) pre-miRNA is exported to cytoplasm

4) pre-miRNA is acted on by Dicer to produce mature miRNA (need Dicer so its functional)

5) mature miRNA is cleaved by RNA helicase (one strand)

6) mature miRNA is incoportaed into protein complexes (RISC) which binds to target mRNA

if complementary to target --> target mRNA is cleaved

if complementary is only partial --> the complex blocks translation of mRNA

What is RNAi?

process of small interfering RNA's (siRNA) binding to the mRNA and silencing it

What is helix turn helix?

20 A.A in 2 short a helical (each 7-9 residues) separated by a B turn

- one of the 2 a helical segments is the recogniiton helix which interacts with the DNA

What is the Zinc finger motif?

30 A.A. residues form loop held by one Zn 2+ ion which is connected to 4 residues (4 cys, or 2 cys and 2 His)

Zinc does not interact with DNA, it just stabilizes the motif

What is a homeodomain?

a part of a protein that binds to DNA and switches other genes on or off (functions as transcription regulator)

- discovered in genes that regulate body patterns

- related to helix-turn-helix motif

What is helix-loop-helix?

implicated in control of gene expression, 2 short ampipathic a helices linked by loop of variable length

one helix is for recognition and the other helix is for forming a dimer with a 3rd helix

DNA binding region with basic residues

What is a leucine zipper?

ampipathic a helic with serious of hydrophobic amino acid residues on one side --> Leu residues at every 7th position

when the 2 helices join --> they form a zipper

DNA binding region that has Lys and Arg (+) residues that can bind negative DNA

What is the sigma factor only in?

prokaryotes

What includes 2 conserved sequences at -35 and -10?

prokaryotes

What has general transcription factors and TATA box?

eukaryotes

What do both eukaryotes and prokaryotes have?

sequences downstream from transcription start site

highly conserved DNA sequences

Does a general transcription factor initiate or increase transcription?

initiate

When does splicing occur?

When the mRNA is still in the nucleus

What is the major state of chromatin found?

euchromatin in prokaryotes

What are negative and positive regulation?

neg= with repressor proteins

pos= with activator proteins

2 diff scenarios for each (KNOW)