Bio Lecture 5 (chapter 6) (DNA TO RNA)

What are "sense" RNA strands?

""sense" strands encode nucleotides that specify amino acids of gene products

What are "anti-sense" RNA strands?

"anti-sense" strands are complementary to "sense" strands

How many RNA polymerases are used in eukaryotes?

3.

Eukaryotic mRNA undergoes several types of processing, what are they?

Removal of introns 5' capping RNA splicing 3' polyadenylation

What is a spliceosome?

large and complex RNA/protein structure that carries out splicing. More than 40 proteins, and numerous small RNAs called snRNAs (small, nuclear RNAs).

Where does translation occur?

occurs on the ribosome. the ribosomes are outside of the nucleus attached to rough ER.

The removal of each intron must be very precise because...

one base addition/deletion will make a huge difference

What are the differences in RNA synthesis vs DNA synthesis?

1.) ribonucleoside triphosphates are monomers 2.)Only one strand of DNA is template for RNA 3.) No primer required, de novo synthesis 4.) RNA is unstable, short-lived compared to DNA 5.) Occurs in the nucleus

Termination for RNA pol 1:

18 nuc. Sequence Recognized by terminator protein

What are the differences between prokaryotic and eukaryotic RNA?

7-Methyl guanosine cap on 5' end Poly-A tail on 3' end Splicing out introns in eukaryotes, the primary transcripts or pre-mRNAs often must be processed by the excision of introns and the addition of 5'7-methyl guanosine caps (MG) and 3'poly(a) tails [(A)n] (Slide 15)

What is the conserved sequence in termination?

AAUAAA

What are the differences between Bacterial and eukaryotic transcription?

Bacteria RNA pol requires a single additional protein for transcription initiation(sigma factor), whereas eukaryotic RNA pol require many additional proteins (called general transcription factors). AND Eukaryotic transcription initiation must deal with packing of DNA into nucleosomes & higher order forms of chromatin structure; these features are absent in bacterial chromosomes

What does the binding of TFIID do to the DNA of the TATA box?

Causes a distortion/bend.

Where does cleavage occur in termination?

Cleavage occurs 11-30 nucleotides downstream from conserved seq.

When are introns "spliced out?"

During eukaryotic pre-mRNA processing

Are all genes expressed with the same efficiencies?

NO, some genes can be expressed more. For example, gene A can be transcribed and translated much more efficiently than gene B. This allows the amount of protein A in the cell to be much greater than that of protein B.

he transcripts of many eucaryotic genes (estimated at 75% of genes in humans) are spliced in more than one way, thereby allowing the same gene to produce a corresponding set of different proteins (Figure 6-27). Rather than being the wasteful process it may have seemed at first sight, RNA splicing enables eucaryotes to increase the already enormous coding potential of their genomes.

Fig. 13-1: The excision of intron sequences from primary transcripts by RNA splicing. The splicing mechanism must be accurate to the single nucleotide to assure that codons in downstream exons are translated correctly to produce the right amino acid sequence in the polypeptide product.

Steps in pre-mRNA splicing:

First step, the snRNPs interact with intronic sequences and each other to cleave the 5' exon/intron junction -U1 binds to 5' intron consensus sequence Second step, lariat formation -U2 snRNP binds to the TACTAAC box, uses A to form lariat the 3' intron/exon junction is cleaved formation of phophodiester bond between exons -U5 snRNP binds to 3' site, U4/U6 snRNP bind

Spliceosome assembly:

First step, the snRNPs themselves interact with intronic sequences and each other to cleave the 5' exon/intron junction Second step, the 3' intron/exon junction is cleaved to release the lariat-shaped intron accompanied by ligation of the exons

Why is phosphorylation of the Pol 2 tail required?

For escape from pre initiation complex (begins elongation)

What is the spliceosome assembled from?

Four snRNPs. Contains: each is complexed with at least seven protein subunits to form a snRNP (small nuclear ribonucleoprotein)

What is required for initiation of transcription via RNA Pol 2?

General Transcription Factors

What does the initiation of transcription require?

Help from proteins called Transcription factors, Binding of Polymerase complex to specific site, and Unwinding of DNA

What are transcriptional activators?

In Eukaryotes, gene regulatory proteins that must bind to specific sequences in DNA and help to attract RNA polymerase II to the start point of transcription

Where is mRNA synthesized in eukaryotes?

In the Nucleus.

Where are proteins synthesized in eukaryotes?

In the cytoplasm.

First step in pre-mRNA splicing:

In the first step, a specific adenine nucleotide in the intron sequence (indicated in red) attacks the 5' splice site and cuts the sugar- phosphate backbone of the RNA at this point. The cut 5' end of the intron becomes covalently linked to the adenine nucleotide

Where are the primary transcripts in eukaryotic transcription? What happens to them?

In the nucleus, they are called heterogeneous nuclear RNA (hnRNA). They are transported to cytoplasm and coated with RNA binding protein for protection from RNases

Why is the 7-methyl guanosine cap made?

It protects the 5' from degradation and it is recognized by translation machinery.

What kinds of RNA can be formed via transcription?

Messenger RNA (mRNA) Transfer RNA (tRNA) Small nuclear RNA (snRNA) Ribosomal RNA (rRNA) Micro RNA (miRNA)

Model of the structure of RNA polymerase II in the yeast S. cerevisiae:

Model of the structure of RNA polymerase II in the yeast S. cerevisiae. Surface grooves on the enzyme are thought to be binding sites for DNA and the growing RNA chain. One groove about 2.5 nm wide and 1nm deep is the putative binding site for DNA (the ten-beads-long chain), another grove about 1.5 nm wide and 2 nm deep could bind the growing RNA chain (the four-beads-long chain).

Eukaryotic genes are interrupted, but prokaryotic genes aren't. Why?

Prokaryotes don't have introns! Prokaryotic genes have a continuous sequence of nucleotide (specify a colinear amino acid sequence of protein chain)

Is prokaryotic transcription/translation separated by cellular compartments?

Nah bro, its not. They usually occur simultaneously/coupled.

Is a primer required for Elongation by RNA pol 2?

No

Is the process of elongation by RNA Pol 2 different in eukaryotes vs prokaryotes?

No, it is the same process.

Where is Eukaryotic RNA Polymerase 1 found and what are its products?

Nucleolus, rRNA, not 5S rRNA

Something about RNA polymerase I don't get...

Nucleophilic attack by 3'OH on the nucleotidyl phosphorus atom with the elimination of pyrophosphate

Where is Eukaryotic RNA Polymerase 2 found and what are its products?

Nucleus, Pre-mRNAs, miRNAs

Where is Eukaryotic RNA Polymerase 3 found and what are its products?

Nucleus, tRNA 5S rRNA and snRNAs

During RNA synthesis how many DNA strands are used as templates? Which one(s)?

Only one DNA strand of a gene is used as a template, and that strand is the NONTEMPLATE strand. (slide 8 for picture)

In RNA Pol 2 termination, what happens to the 3'?

Poly (A) polymerase adds about 200 adenines to it. Cleavage and polyadenylation are coupled.

Structure of Eukaryotic mRNA Molecule:

Pre-mRNAs modified at 5' end 7-methyl guanosine (7-MG) cap Unusual 5'-5' triphosphate bond

What is the first modification of eukaryotic pre-mRNAs?

RNA Capping; As soon as RNA polymerase II has produced about 25 nucleotides of RNA, the 5' end of the new RNA molecule is modified by addition of a cap that consists of a modified guanine nucleotide

Where does RNA synthesis occur?

RNA synthesis occurs within a locally unwound segment of DNA. (slide 11)

Just remember this:

Signal Sequence for splicing: -dinucleotide 5'-Exon-[GT---INTRON---AG]-Exon-3' -consensus sequences 5'--exon -[GTAAGT......(6Py)NCAG]-exon--3' -Intron internal signal sequence TACTAAC box is 30 nuc upstream from 3' splice site

Termination for RNA pol 3

Similar to rho-independent mechanism of prokaryotes

Is the RNA produced by transcription single or double stranded?

Single

Picture of RNA polymerase:

Slide 10

What is the structure of the promoter recognized by RNA Polymerase 2?

TATA Box, The assembly process begins when the general transcription factor TFIID binds to a short double-helical DNA sequence primarily composed of T and A nucleotides.

The RNA produced by transcription is complementary to the DNA _____________ strand.

Template

What recruits RNA processing enzymes?

The "tail" of RNA Pol

How are the 5' and 3' ends of mRNA molecules produced in prokaryotes?

The 5' end of an mRNA molecule is produced by the initiation of transcription, and the 3' end is produced by the termination of transcription.

Steps taken by RNA Polymerase:

The RNA polymerase (pale blue) moves stepwise along the DNA, unwinding the DNA helix at its active site. As it progresses, the polymerase adds nucleotides (represented as small "T" shapes) one by one to the RNA chain at the polymerization site, using an exposed DNA strand as a template. The RNA transcript is thus a complementary copy of one of the two DNA strands. A short region of DNA/RNA helix (approximately nine nucleotide pairs in length) is therefore formed only transiently, and a "window" of DNA/RNA helix therefore moves along the DNA with the polymerase. The incoming nucleotides are in the form of ribonucleoside triphosphates (ATP, UTP, CTP, and GTP), and the energy stored in their phosphate-phosphate bonds provides the driving force for the polymerization reaction

What are the protein cofactors required for initiation?

Transcription factors

What is the flow of genetic information?

The flow of genetic information from DNA to RNA (transcription) and from RNA to protein (translation) occurs in all living cells.

What do general transcription factors do?

The general transcription factors help to position eucaryotic RNA polymerase correctly at the promoter, aid in pulling apart the two strands of DNA to allow transcription to begin, and release RNA polymerase from the promoter into the elongation mode once transcription has begun.

How are intron sequences removed?

The intron sequences are removed from the newly synthesized RNA through the process of RNA splicing. Each splicing event removes one intron, proceeding through two sequential phosphoryl-transfer reactions known as transesterifications; these join two exons while removing the intron as a "lariat."

Where is ribosomal (r)RNA and ribosome synthesis?

The nucleolus

Human Beta-globin gene and human Factor VIII gene:

The relatively small b-globin gene, which encodes one of the subunits of the oxygen-carrying protein hemoglobin, contains 3 exons (see also Figure 4-7). (B) The much larger Factor VIII gene contains 26 exons; it codes for a protein (Factor VIII) that functions in the blood-clotting pathway. The most prevalent form of hemophilia results from mutations in this gene.

How does RNA polymerase elongate the RNA chain?

The triphosphate group from Uracil is added to the 2 OH group of guanine, with chain growth occurring in the 5' to the 3'.

Is bacterial RNA polymerase similar to RNA polymerase 2 of eukaryotes?

They are structurally similar, but there are differences between eukaryotic & prokaryotic transcription

How is the RNA capping reaction performed?

Three enzymes, acting in succession, perform the capping reaction: one (a phosphatase) removes a phosphate from the 5' end of the nascent RNA, another (a guanyl transferase) adds a GMP in a reverse linkage (5¢ to 5¢ instead of 5' to 3'), and a third (a methyl transferase) adds a methyl group to the guanosine

Initiation of transcription of a eucaryotic gene by RNA polymerase II.

To begin transcription, RNA polymerase requires several general transcription factors. (A) The promoter contains a DNA sequence called the TATA box, which is located 25 nucleotides away from the site at which transcription is initiated. (B) Through its subunit TBP, TFIID recognizes and binds the TATA box, which then enables the adjacent binding of TFIIB (C). For simplicity the DNA distortion produced by the binding of TFIID (see Figure 6-18) is not shown. (D) The rest of the general transcription factors, as well as the RNA polymerase itself, assemble at the promoter. (E) TFIIH then uses ATP to pry apart the DNA double helix at the transcription start point, locally exposing the template strand. TFIIH also phosphorylates RNA polymerase II, changing its conformation so that the polymerase is released from the general factors and can begin the elongation phase of transcription.

What happens in transcription?

Transfer of genetic information from DNA to RNA

True or False: Transcription & translation occur in different compartments in eukaryotes

True

True or False: Transcription of DNA yields a single stranded RNA that is complementary to one strand of DNA.

True

True or False: transcription initiation in a eucaryotic cell typically requires the local recruitment of chromatin modifying enzymes, including chromatin remodeling complexes and histone- modifying enzymes.

True

True or False; The LARGER the s value, the LARGER the rRNA.

True.

What are the steps in pre-mRNA splicing?

Two steps: Cleavage and Ligation

What is a gene?

Unit of genetic information that controls the synthesis of one protein or structural RNA molecule

Alternative splicing of some eukaryotic mRNAs gives rise to _______ __________.

Variant Proteins

What does the Phosphorylation state of the RNA tail determine?

Which factors are recruited!

Where does the extension of the RNA chain in elongation occur?

Within the transcription bubble.

Is eukaryotic RNA synthesis more complex vs prokaryotic?

Yes.

Do eukaryotic genes have both eons and introns?

Yes. Eukaryotic genes have both expressed (exon) sequences & non-expressed (introns) sequences

Is a short RNA/DNA heteroduplex formed in Elongation by RNA pol 2?

Yesssir.

The DNA bending caused by TF can serve as....

a "recruitment site" for other proteins that take part in transcription. This distortion is thought to serve as a physical landmark for the location of an active promoter in the midst of a very large genome, and it brings DNA sequences on both sides of the distortion together to allow for subsequent protein assembly steps. Other factors then assemble, along with RNA polymerase II, to form a complete transcription initiation complex

What does it mean when we say prokaryotic mRNA is polycistronic?

bacterial mRNAs can contain the instructions for several different proteins, whereas eucaryotic mRNAs nearly always contain the information for only a single protein. Polycistronic: single mRNA can code for several gene

How is a consensus nucleotide sequence derived?

by comparing many sequences with the same basic func- tion and tallying up the most common nucleotide found at each position.

What are consensus sequences?

comparison of many different bacterial promoters reveals a surprising degree of variation. Nevertheless, they all contain related sequences, reflecting in part aspects of the DNA that are recognized directly by the sigma factor. These common features are often summarized in the form of a consensus sequence .

How are general transcription factors denoted?

denoted "TFII-X" (TFII = TF for Pol II)

What do exons encode?

different functional domains

Where does snRNA exist?

each is complexed with at least seven protein subunits to form a snRNP (small nuclear ribonucleoprotein)

What is the transcript 3' end produced by in RNA Pol 2 termination?

endonucleolytic cleavage.

In what direction does RNA transcription occur?

in the 5' to 3' direction.

What does polyadenlation do?

increases stability.

Eukaryotes synthesize _____________ mRNAs.

monogenic

Prokaryotes synthesize _____________ mRNAs.

multigenic

The consensus nucleotide sequences in an RNA molecule that signal the beginning and the end of most introns in humans.

nly the three blocks of nucleotide sequences shown are required to remove an intron sequence; the rest of the intron can be occupied by any nucleotides. Here A, G, U, and C are the standard RNA nucleotides; R stands for purines (A or G); and Y stands for pyrimidines (C or U).

What is an intron?

noncoding, intervening sequences found in eukaryotes.

The RNA produced by transcription is IDENTICAL to the DNA _____________ strand.

nontemplate (T's replaced by U)

What is exon shuffling?

rearrangement of eons, leading to the evolution of "new" exon combinations.

What is the transcriptional unit?

segment of DNA transcribed to produce one RNA molecule can be one or several genes

What is an exon?

sequences that remain in mature mRNA. Coding and non-coding

Image of Eukaryotic RNA synthesis:

slide 14

What is the direction of transcription determined by?

the promoter at the beginning of each gene.

What is the advantage of RNA splicing?

the transcripts of many eucaryotic genes (estimated at 75% of genes in humans) are spliced in more than one way, thereby allowing the same gene to produce a corresponding set of different proteins. Rather than being the wasteful process it may have seemed at first sight, RNA splicing enables eucaryotes to increase the already enormous coding potential of their genomes.

What do RNA polymerases do?

they catalyzes the synthesis of an RNA complement to DNA. catalyzes the formation of the phosphodiester bonds that link the nucleotides together to form a linear chain. Unwinds and rewinds DNA RNA chain becomes elongated.

Which strand of DNA does transcription occur on?

transcription can occur off of either strand of DNA

Where does transcription occur?

transcription occurs in the nucleus of a cell, if it has a nucleus. If the cell lacks a nucleus, then the mRNA is immediately translated without additional processing.

What is a mediator?

ucaryotic transcription initia- tion in vivo requires the presence of a protein complex known as Mediator, which allows the activator proteins to communicate properly with the poly- merase II and with the general transcription factors.

What does RNA Pol do in elongation?

unwinds and winds DNA.

When does the 7-methyl guanosine cap occur?

when the chain is about 30 nucleotides in length (during active transcription)

The cut 5' end of the intron becomes covalently linked to the adenine nucleotide:

yeah....